Gene variants coding for proteins from the metabolic pathway of fine chemicals

ABSTRACT

The present invention relates to mutated nucleic acids and proteins of the metabolic pathway of fine chemicals, to processes for preparing genetically modified producer organisms, to processes for preparing fine chemicals by culturing genetically modified organisms, and to genetically modified organisms themselves.

RELATED APPLICATIONS

The present application is a 35 U.S.C. 371 national stage filing ofInternational Application No. PCT/EP2004/014338, filed Dec. 16, 2004,which claims priority to German Application No. 10359661.5, filed Dec.18, 2003. The entire contents of each of these applications are herebyincorporated by reference herein.

The present invention relates to mutated nucleic acids and proteins ofthe metabolic pathway of fine chemicals, to processes for preparinggenetically modified producer organisms, to processes for preparing finechemicals by culturing said genetically modified organisms and to saidgenetically modified organisms themselves.

Many products and byproducts of naturally occurring metabolic processesin cells are used in many branches of industry, including the foodindustry, the animal feed industry, the cosmetic industry and thepharmaceutical industry. These compounds, which are collectivelyreferred to as “fine chemicals”, comprise, for example, organic acids,both proteinogenic and nonproteinoge amino acids, nucleotides andnucleosides, lipids and fatty acids, diols, carbohydrates, aromaticcompounds, vitamins and cofactors and also enzymes.

They may be produced, for example, by large-scale fermentation ofmicroorganisms which have been developed to produce and secrete largeamounts of one or more desired fine chemicals.

An organism which is particularly suitable for this purpose isCorynebacterium glutamicum, a Gram-positive, nonpathogenic bacterium.Using strain selection, a number of mutant strains have been developedwhich produce various desirable compounds. The selection of strainswhich have been improved with respect to production of a particularcompound is, however, a time-consuming and difficult process.

It is possible to increase the productivity of producer organisms bygenetic modifications. For example, the specific mutation of particulargenes in a producer organism may result in an increase in productivityof a desired fine chemical.

EP 1 108 790 A2 describes, starting from the wild-type sequence encodinga Corynebacterium glutamicum homoserine dehydrogenase, a mutated nucleicacid sequence encoding a homoserine dehydrogenase which has, compared tothe wild-type sequence, the mutation Val59Ala. Furthermore, a mutatednucleic acid sequence encoding a pyruvate carboxylase which has themutation Pro458Ser in comparison with the Corynebacterium glutamicumwild-type amino acid sequence is described. Introduction of saidmutations into Corynebacterium glutamicum results in an increase in thelysine yield.

WO 0063388 furthermore discloses a mutated ask gene which encodes anasparto-kinase having the mutation T311I.

Other mutations in genes and proteins of the Corynebacterium glutamicumbiosynthetic pathway of fine chemicals are described in WO 0340681, WO0340357, WO 0340181, WO 0340293, WO 0340292, WO 0340291, WO 0340180, WO0340290, WO 0346123, WO 0340289 and WO 0342389.

Although the mutations known in the prior art already result in producerorganisms having optimized productivity, i.e. optimized yield of thedesired fine chemical and optimized C yield, there is a constant needfor further improving the productivity of said organisms.

It is an object of the present invention to provide further mutatedgenes and proteins which result in an increase in productivity inproducer organisms of fine chemicals and thus in an improvement ofbiotechnological processes for preparing fine chemicals.

We have found that this object is achieved by proteins having thefunction indicated in each case in table 1/column 7 and having an aminoacid sequence which has in at least one of the amino acid positionswhich, starting from the amino acid sequence referred to in each case intable 1/column 2, correspond to the amino acid positions indicated forsaid amino acid sequence in table 1/column 4 a proteinogenic amino aciddifferent from the particular amino acid indicated in the same row intable 1/column 5, with the proviso that the proteins according to table2 are excepted.

The present invention provides novel nucleic acid molecules and proteinswhich, on the one hand, can be used for identifying or classifyingCorynebacterium glutamicum or related bacterial species and, on theother hand, result in an increase in productivity in producer organismsof fine chemicals and thus in an improvement of biotechnologicalprocesses for preparing fine chemicals.

C. glutamicum is a Gram-positive, aerobic bacterium which is widely usedin industry for large-scale production of a number of fine chemicals andalso for the degradation of hydrocarbons (e.g. in the case of crude oilspills) and for the oxidation of terpenoids. The nucleic acid moleculesmay therefore be used furthermore for identifying microorganism whichcan be used for production of fine chemicals, for example byfermentation processes. Although C. glutamicum itself is nonpathogenic,it is, however, related to other Corynebacterium species, such asCorynebacterium diphtheriae (the diphtheria pathogen), which are majorpathogens in humans. The ability to identify the presence ofCorynebacterium species may therefore also be of significant clinicalimportance, for example in diagnostic applications. Moreover, saidnucleic acid molecules may serve as reference points for mapping the C.glutamicum genome or the genomes of related organisms.

The proteins of the invention, also referred to as Metabolic-Pathwayproteins or MP proteins hereinbelow, have the function indicated in eachcase in table 1/column 7. Furthermore, they have in each case an aminoadd sequence which has in at least one of the amino acid positionswhich, starting from the amino acid sequence referred to in each case intable 1/column 2, correspond to the amino acid positions indicated forsaid amino acid sequence in table 1/column 4 a proteinogenic amino aciddifferent from the particular amino acid indicated in the same row intable 1/column 5.

The “corresponding” amino acid position preferably means the amino acidposition of the amino acid sequence of the MP proteins of the invention,which the skilled worker can readily find

-   a) by homology comparison of the amino acid sequence or-   b) by structural comparison of the secondary, tertiary and/or    quaternary structure of said amino acid sequence    with the amino acid sequence referred to in each case in table    1/column 2 and having the amino acid position indicated for said    amino acid sequence in each case in table 1/column 4.

A preferred method of comparing homologies of the amino acid sequencesis employed, for example, by the Lasergene Software from DNASTAR, inc.Madison, Wis. (USA), using the Clustal method (Higgins D G, Sharp P M.Fast and sensitive multiple sequence alignments on a microcomputer.Comput Appl. Biosci. 1989 April; 5(2):151-1), setting the followingparameters:

Multiple Alignment Parameter:

-   Gap penalty 10-   Gap length penalty 10

Pairwise Alignment Parameter:

-   K-tuple 1-   Gap penalty 3-   Window 5-   Diagonals saved 5

In a preferred embodiment, the proteins have the function indicated ineach case in table 1/column 7 and an amino acid sequence which has in anamino acid position which, starting from the amino acid sequencereferred to in each case in table 1/column 2, corresponds to the aminoacid position indicated for said amino acid sequence in table 1/column 4a proteinogenic amino acid different from the particular amino acidindicated in the same row in table 1/column 5, with the proviso that themutated proteins according to table 2 are excepted.

In a further preferred embodiment, the proteins of the invention havethe amino acid sequence referred to in each case in table 1/column 2,where said protein has in at least one of the amino acid positionsindicated for said amino acid sequence in table 1/column 4 aproteinogenic amino acid different from the particular amino acidindicated in the same row in table 1/column 5.

In a further preferred embodiment, the proteins of the invention havethe amino acid sequence referred to in each case in table 1/column 2,where said protein has in one of the amino acid positions indicated forsaid amino acid sequence in table 1/column 4 a proteinogenic amino aciddifferent from the particular amino acid indicated in the same row intable 1/column 5.

The amino acid sequences indicated in table 1/column 2 areCorynebacterium glutamicum wild-type sequences. Table 1/column 4indicates for the particular wild-type amino acid sequence at least oneamino acid position in which the amino acid sequence of the proteins ofthe invention has a proteinogenic amino acid different from theparticular amino acid indicated in the same row in table 1/column 5.

In a further preferred embodiment, the proteins have in at least one ofthe amino acid positions indicated for the amino acid sequence in table1/column 4 the amino acid indicated in the same row in table 1/column 6.

Another aspect of the invention relates to an isolated MP protein or toa section thereof, for example a biologically active section thereof. Ina preferred embodiment, the isolated MP protein or its section regulatestranscriptionally, translationally or posttranslationally one or moremetabolic pathways in organisms, in particular in corynebacteria andbrevibacteria.

The MP polypeptide or a biologically active section thereof may befunctionally linked to a non-MP polypeptide to produce a fusion protein.In preferred embodiments, the activity of this fusion protein isdifferent from that of the MP protein alone and, in other preferredembodiments, said fusion protein regulates transcriptionally,translationally or posttranslationally one or more metabolic pathways inorganisms, in particular in corynebacteria and brevibacteria, preferablyin Corynebacterium glutamicum. In particularly preferred embodiments,integration of said fusion protein into a host cell modulates productionof a compound of interest by the cell.

The invention furthermore relates to isolated nucleic acids encoding anabove-described protein of the invention. These nucleic acids arehereinbelow also referred to as Metabolic-Pathway nucleic acids or MPnucleic acids or MP genes. These novel MP nucleic acid molecules encodethe MP proteins of the invention. These MP proteins may, for example,exert a function which is involved in the transcriptional, translationalor posttranslational regulation of proteins which are crucial for thenormal metabolic functioning of cells. Owing to the availability ofcloning vectors for use in Corynebacterium glutamicum, as disclosed, forexample, in Sinskey et al., U.S. Pat. No. 4,649,119, and of techniquesfor the genetic manipulation of C. glutamicum and the relatedBrevibacteium species (e.g. lactofermentum) (Yoshihama et al., J.Bacteriol. 162 (1985) 591-597; Katsumata et al., J. Bacteriol. 159(1984) 306-311; and Santamaria et al. J. Gen. Microbiol. 130 (1984)2237-2246), the nucleic acid molecules of the invention can be used forgenetic manipulation of said organism in order to make it a better andmore efficient producer of one or more fine chemicals.

A suitable starting point for preparing the nucleic acid sequences ofthe invention is, for example, the genome of a Corynebacteriumglutamicum strain which is obtainable under the name ATCC 13032 from theAmerican Type Culture Collection.

Customary methods can be employed for preparing from these nucleic acidsequences the nucleic acid sequences of the invention, using themodifications listed in table 1. It is advantageous to use forback-translation of the amino acid sequence of the MP proteins of theinvention into the inventive nucleic acid sequences of the MP genes thecodon usage of that organism into which the MP nucleic acid sequence ofthe invention is to be introduced or in which the nucleic acid sequenceof the invention is present. For example, it is advantageous to use thecodon usage of Corynebacterium glutamicum for Corynebacteriumglutamicum. The codon usage of the particular organism can be determinedin a manner known per se from databases or patent applicationsdescribing at least one protein and one gene encoding this protein ofthe organism of interest.

An isolated nucleic acid molecule encoding an MP protein can begenerated by introducing one or more nucleotide substitutions, additionsor deletions into a nucleotide sequence of table 1/column 1 so that oneor more amino acid substitutions, additions or deletions are introducedinto the encoded protein. Mutations can be introduced into one of thesequences of table 1/column 1 by standard techniques such assite-specific mutagenesis and PCR-mediated mutagenesis. Preferably,conservative amino acid substitutions are introduced at one or more ofthe predicted nonessential amino acid residues. A “conservative aminoacid substitution” is one in which the amino acid residue is replaced byan amino acid residue having a similar side chain. Families of aminoacid residues having similar side chains have been defined in the art.These families include amino acids having basic side chains (e.g.lysine, arginine, histidine), acidic side chains (e.g. aspartic acid,glutamic acid), uncharged polar side chains (e.g. glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g. threonine,valine, isoleucine) and aromatic side chains (e.g. tyrosine,phenylalanine, tryptophan, histidine). A predicted nonessential aminoacid residue in an MP protein is thus preferably replaced by anotheramino acid residue of the same side-chain family. Alternatively, inanother embodiment, the mutations can be introduced randomly along allor part of the MP-encoding sequence, for example by saturationmutagenesis, and the resulting mutants can be tested for the MP activitydescribed herein, in order to identify mutants which retain an MPactivity. After mutagenesis of one or the sequences of Appendix A, theencoded protein can be expressed recombinantly, and the activity of saidprotein can be determined, for example, using the assays describedherein (see example 8 of the Examples section).

The present invention is based on making available novel molecules whichare referred to herein as MP nucleic acid and MP protein molecules andwhich regulate one or more metabolic pathways in organisms, inparticular in corynebacteria or brevibacteria, particularly preferablyin C. glutamicum, by transcriptional, translational or posttranslationalmeasures. In one embodiment, the MP molecules regulatetranscriptionally, translationally or posttranslationally one metabolicpathway in organisms, in particular in corynebacteria or brevibacteria,particularly preferably in C. glutamicum. In a preferred embodiment, theactivity of the inventive MP molecules for regulating one or moremetabolic pathways in organisms, in particular in corynebacteria orbrevibacteria, particularly preferably in C. glutamicum, has an effecton the production of a fine chemical of interest by said organism. In aparticularly preferred embodiment, the MP molecules of the inventionhave a modulated activity so that the metabolic pathways of organisms,in particular in corynebacteria or brevibacteria, particularlypreferably in C. glutamicum, which pathways are regulated by the MPproteins of the invention, are modulated with respect to theirefficiency or their throughput and this modulates either directly orindirectly the yield, production and/or efficiency of production of afine chemical of interest by organisms, in particular in corynebacteriaor brevibacteria, particularly preferably in C. glutamicum.

The term “MP protein” or “MP polypeptide” comprises proteins whichregulate transcriptionally, translationally or posttranslationally ametabolic pathway in organisms, in particular in corynebacteria orbrevibacteria, particularly preferably in C. glutamicum. Examples of MPproteins comprise those listed in table 1. The terms “MP gene” and “MPnucleic acid sequence” comprise nucleic acid sequences encoding an MPprotein which comprises a coding region and corresponding untranslated5′ and 3′ sequence regions. Examples of MP genes are listed in table 1.

The terms “production” and “productivity” are known in the art andinclude the concentration of the fermentation product (for example ofthe fine chemical of interest) produced within a predetermined timeinterval and a predetermined fermentation volume (e.g. kg of product perh per l).

The term “efficiency of production” comprises the time required forattaining a particular production quantity (for example the timerequired by the cell for reaching a particular throughput rate of a finechemical). The term “yield” or “product/carbon yield” is known in theart and comprises the efficiency of converting the carbon source intothe product (i.e. the fine chemical). This is, for example, usuallyexpressed as kg of product per kg of carbon source. Increasing the yieldor production of the compound increases the amount of the moleculesobtained or of the suitable obtained molecules of this compound in aparticular culture volume over a predetermined period.

The terms “biosynthesis” and “biosynthetic pathway” are known in the artand comprise the synthesis of a compound, preferably an organiccompound, from intermediates by a cell, for example in a multistepprocess or highly regulated process. The terms “degradation” and“degradation pathway” are known in the art and comprise cleavage of acompound, preferably an organic compound, into degradation products (inmore general terms: smaller or less complex molecules) by a cell, forexample in a multistep process or highly regulated process.

The term “metabolism” is known in the art and comprises the entirety ofbiochemical reactions which take place in an organism. The metabolism ofa particular compound (e.g. the metabolism of an amino acid such asglycine) then comprises all biosynthetic, modification and degradationpathways of this compound in the cell.

The term “regulation” is known in the art and comprises the activity ofa protein for controlling the activity of another protein. The term“transcriptional regulation” is known in the art and comprises theactivity of a protein for inhibiting or activating the conversion of aDNA encoding a target protein into mRNA. The term “translationalregulation” is known in the art and comprises the activity of a proteinfor inhibiting or activating conversion of an mRNA encoding a targetprotein into a protein molecule. The term “posttranslational regulation”is known in the art and comprises the activity of a protein forinhibiting or improving the activity of a target protein by covalentlymodifying said target protein (e.g. by methylation, glycosylation orphosphorylation).

The improved yield, production and/or efficiency of production of a finechemical may be caused directly or indirectly by manipulating a gene ofthe invention. More specifically, modifications in MP proteins whichusually regulate the yield, production and/or efficiency of productionof a fine chemical of a fine-chemical metabolic pathway may have adirect effect on the total production or the production rate of one ormore of these desired compounds from this organism.

Modifications in those proteins which are involved in these metabolicpathways may also have an indirect effect on the yield, productionand/or efficiency of production of a fine chemical of interest.Metabolism regulation is inevitably complex and the regulatorymechanisms which effect the different pathways may overlap in manyplaces so that more than one metabolic pathway can be adjusted quicklyaccording to a particular cellular event. This makes it possible for themodification of a regulatory protein for one metabolic pathway to alsoaffect many other metabolic pathways, some of which may be involved inthe biosynthesis or degradation of a fine chemical of interest. In thisindirect manner, modulation of the action of an MP protein may have aneffect on the production of a fine chemical which is produced via ametabolic pathway different from that directly regulated by said MPprotein.

The MP nucleic acid and MP protein molecules of the invention may beused in order to directly improve the yield, production and/orefficiency of production of one or more fine chemicals of interest fromnonhuman organisms.

It is possible, by means of gene recombination techniques known in theart, to manipulate one or more regulatory proteins of the invention soas for their function to be modulated. The mutation of an MP proteinwhich is involved in repressing the transcription of a gene encoding anenzyme required for the biosynthesis of an amino acid, such that saidamino acid is no longer capable of repressing said transcription, maycause, for example, an increase in production of said amino acid.

Accordingly, modification of the activity of an MP protein, which causesan increased translation or activates posttranslational modification ofan MP protein involved in the biosynthesis of a fine chemical ofinterest, may in turn increase production of said chemical. The oppositesituation may likewise be useful: by increasing the repression oftranscription or translation or by posttranslational negativemodification of an MP protein involved in regulating the degradationpathway of a compound, it is possible to increase production of saidchemical. In any case, the total yield or the production rate of thefine chemical of interest may be increased.

Likewise, it is possible that said modifications in the protein andnucleotide molecules of the invention may improve the yield, productionand/or efficiency of production of fine chemicals via indirectmechanisms. The metabolism of a particular compound is inevitably linkedto other biosynthetic and degradation pathways in the cell and necessarycofactors, intermediates or substrates in a metabolic pathway areprobably provided or limited by another metabolic pathway. Modulatingone or more regulatory proteins of the invention can therefore influencethe efficiency of the activity of other biosynthetic or degradationpathways of fine chemicals. In addition to this, manipulation of one ormore regulatory proteins may increase the overall ability of the cell togrow and to propagate in culture, particularly in large-scalefermentation cultures in which growth conditions may be suboptimal. Itis possible to increase the biosynthesis of nucleotides and possiblycell division, for example by mutating further an inventive MP proteinwhich usually a repression of the biosynthesis of nucleosides as aresponse to a suboptimal extracellular supply of nutrients (therebypreventing cell division), such that said protein has a lower repressoractivity. Modifications in those MP proteins which cause increased cellgrowth and increased division in culture may cause an increase in theyield, production and/or efficiency of production of one or more finechemicals of interest from the culture, at least owing to the increasednumber of cells producing said chemical in culture.

The invention provides novel nucleic acid molecules encoding proteinsthat are capable of carrying out an enzymic step involved in thetranscriptional, translational or posttranslational regulation ofmetabolic pathways in nonhuman organisms. Nucleic acid molecules whichencode an MP protein are referred to herein as MP nucleic acidmolecules. In a preferred embodiment, the MP protein is involved in thetranscriptional, translational or posttranslational regulation of one ormore metabolic pathways. Examples of such proteins are those encoded bythe genes listed in table 1.

Consequently, one aspect of the invention relates to isolated nucleicacid molecules (e.g. cDNAs) comprising a nucleotide sequence whichencodes an MP protein or biologically active sections thereof and alsonucleic acid fragments which are suitable as primers or hybridizationprobes for detecting or amplifying MP-encoding nucleic acid (e.g. DNA ormRNA). In other preferred embodiments, the isolated nucleic acidmolecule encodes any one of the amino acid sequences listed in table 1.The preferred MP proteins of the invention likewise have preferably atleast one of the MP activities described herein.

In a further embodiment, the isolated nucleic acid molecule is at least15 nucleotides in length and hybridizes under stringent conditions to anucleic acid molecule of the invention. The isolated nucleic acidmolecule preferably corresponds to a naturally occuning nucleic acidmolecule. The isolated nucleic acid more preferably encodes a naturallyoccurring C. glutamicum MP protein or a biologically active sectionthereof.

All living cells have complex catabolic and anabolic capabilities withmany metabolic pathways linked to one another. In order to maintain anequilibrium between various parts of this extremely complex metabolicnetwork, the cell employs a finely tuned regulatory network. Byregulating the enzyme synthesis and enzyme activity, eitherindependently or simultaneously, the cell can regulate the activity ofcompletely different metabolic pathways so as to meet the cell'schanging needs.

The induction or repression of enzyme synthesis may take place either atthe transcriptional or the translational level or at both levels (for areview, see Lewin, B. (1990) Genes IV, Part 3: “Controlling prokaryoticgenes by transcription”, Oxford University Press, Oxford, pp. 213-301,and the references therein, and Michal, G. (1999) Biochemical Pathways:An Atlas of Biochemistry and Molecular Biology, John Wiley & Sons). Allof these known regulatory processes are mediated by additional geneswhich themselves react to various external influences (e.g. temperature,nutrient supply or light). Examples of protein factors involved in thistype of regulation include the transcription factors. These are proteinswhich bind to the DNA, thereby causing expression of a gene either toincrease (positive regulation as in the case of the E. coli ara operon)or to decrease (negative regulation as in the case of the E. coli lacoperon). These expression-modulating transcription factors maythemselves be subject to regulation. Their activity may be regulated,for example, by low molecular weight compounds binding to theDNA-binding protein, whereby binding of said proteins to the appropriatebinding site on the DNA is stimulated (as in the case of arabinose forthe ara operon) or inhibited (as in the case of lactose for the lacoperon) (see, for example, Helmann, J. D. and Chamberlin, M. J. (1988)“Structure and function of bacterial sigma factors” Ann. Rev. Biochem.57: 839-872; Adhya, S. (1995) “The lac and gal operons today” and Boos,W. et al., “The maltose system”, both in Regulation of Gene Expressionin Escherichia coli (Lin, E. C. C. and Lynch, A. S. Editors) Chapman &Hall: New York, pp. 181-200 and 201-229; and Moran, C. P. (1993) “RNApolymerase and transcription factors” in: Bacillus subtilis and othergram-positive bacteria, Sonenshein, A. L. Editor ASM: Washington, D.C.pp. 653-667).

Protein synthesis is regulated not only at the transcriptional level butoften also at the translational level. This regulation can be carriedout via many mechanisms, including modification of the ability of theribosome to bind to one or more mRNAs, binding of the ribosome to mRNA,maintaining or removing of the mRNA secondary structure, the use ofcommon or less common codons for a particular gene, the degree ofabundance of one or more tRNAs and specific regulatory mechanisms suchas attenuation (see Vellanoweth, R. I. (1993) Translation and itsregulation in Bacillus subtilis and other gram-positive bacteria,Sonenshein, A. L. et al. Editor ASM: Washington, D.C., pp. 699-711 andreferences therein).

The transcriptional and translational regulation may be directed towarda single protein (sequential regulation) or simultaneously toward aplurality of proteins in various metabolic pathways (coordinatedregulation). Genes whose expression is regulated in a coordinatedfashion are located in the genome often in close proximity in an operonor regulation. This up or down regulation of gene transcription and genetranslation is controlled by the cellular or extracellular amounts ofvarious factors such as substrates (precursors and intermediates whichare used in one or more metabolic pathways), catabolites (moleculesproduced by biochemical metabolic pathways associated with energyproduction from the degradation of complex organic molecules such assugars) and end products (molecules which are obtained at the end of ametabolic pathway). Expression of genes which encode enzymes requiredfor the activity of a particular metabolic pathway is induced by largeamounts of substrate molecules for said metabolic pathway.Correspondingly, said gene expression is repressed by the presence oflarge intracellular amounts of the end product of the pathway (Snyder,L. and Champness, W. (1977) The Molecular Biology of Bacteria ASM:Washington). Gene expression may likewise be regulated by other externaland internal factors such as environmental conditions (e.g. heat,oxidative stress or hunger). These global environmental changes causechanges in expression of specialized modulating genes which trigger geneexpression directly or indirectly (via additional genes or proteins) bybinding to DNA and thereby induce or repress transcription (see, forexample, Lin, E. C. C. and Lynch, A. S. Editors (1995) Regulation ofGene Expression in Escherichia coli, Chapman & Hall: New York).

Another mechanism by which the cellular metabolism can be regulatedtakes place at the protein level. This regulation is carried out eithervia the activities of other enzymes or via binding of low molecularweight components which prevent or enable normal function of theprotein. Examples of protein regulation by binding of low molecularweight compounds include the binding of GTP or NAD. The binding of lowmolecular weight chemicals is usually reversible, for example in thecase of GTP-binding proteins. These proteins occur in two states (withbound GTP or GDP), with one state being the active form of the proteinand the other one the inactive form.

The protein activity is regulated by the action of other enzymes usuallyvia covalent modification of the protein (i.e. phosphorylation of aminoacid residues such as histidine or aspartate or methylation). Thiscovalent modification is usually reversible and this is effected by anenzyme having the opposite activity. An example of this is the oppositeactivity of kinases and phosphorylases in protein phosphorylation:protein kinases phosphorylate specific residues on a target protein(e.g. serine or threonine), whereas protein phosphorylases remove thephosphate groups from said proteins. Enzymes modulating the activity ofother proteins are usually modulated themselves by external stimuli.These stimuli are mediated by proteins acting as sensors. A well-knownmechanism by which these sensor proteins mediate said external signalsis dimerization, but other mechanisms are also known (see, for example,Msadek, T. et al. (1993) “Two-component Regulatory-Systems” in: Bacillussubtilis and Other Gram-Positive Bacteria, Sonenshein, A. L. et al.,Editors, ASM: Washington, pp. 729-745 and references therein).

A detailed understanding of the regulatory networks which control thecellular metabolism in microorganisms is crucial for the production ofchemicals in high yields by fermentation. Control systems fordown-regulating the metabolic pathways may be removed or reduced inorder to improve the synthesis of chemicals of interest and,correspondingly, those for up-regulating the metabolic pathway of aproduct of interest may be constitutively activated or optimized withrespect to the activity (as shown in Hirose, Y. and Okada, H. (1979)“Microbial Production of Amino Acids”, in: Peppler, H. J. and Perlman,D. (Editors) Microbial Technology 2nd Edition, Vol. 1, Chapter 7,Academic Press, New York).

Another aspect of the invention relates to nucleic acid constructs suchas, for example, vectors such as recombinant expression vectors, forexample, which contain at least one nucleic acid of the invention.

This nucleic acid construct preferably comprises in a functionallylinked manner a promoter and, if appropriate, a terminator. Particularpreference is given to promoters which are heterologous with respect tothe nucleic acid and are capable of expressing said nucleic acid in thenonhuman organisms. An example of a particularly preferred promoter inthe preferred organisms of the genus Corynebacterium or Brevibacteriumis the tac promoter.

The invention further relates to a process for preparing a nonhuman,genetically modified organism by transforming a nonhuman parent organismby introducing into said parent organism

-   a) at least one above-described MP nucleic acid of the invention or-   b) at least one above-described nucleic acid construct of the    invention or-   c) a promoter which is heterologous with respect to the    above-described endogenous MP nucleic acid of the invention and    which enables said endogenous MP nucleic acid of the invention to be    expressed in said organism.

Preferably, the promoter according to embodiment c) is introduced intothe organism so as for the promoter to be functionally linked in saidorganism to the endogenous MP nucleic acid of the invention. A“functional linkage” means a linkage which is functional, i.e. a linkagewhich enables the endogenous MP nucleic acid of the invention to beexpressed by the introduced promoter.

The term “parent organism” means the corresponding nonhuman organismwhich is transformed to the genetically modified organism. A parentorganism here may be a wild-type organism or an organism which hasalready been genetically modified. Furthermore, the parent organisms mayalready be capable of producing the fine chemical of interest or beenabled by the transformation of the invention to produce the finechemical of interest.

The term “genetically modified organism” preferably means a geneticallymodified organism in comparison with the parent organism.

Depending on the context, the term “organism” means the nonhuman parentorganism or a nonhuman, genetically modified organism of the inventionor both.

The MP nucleic acid of the invention or the nucleic acid construct ofthe invention may be introduced chromosomally or plasmidally as aself-replicating plasmid. The MP nucleic acids of the invention or thenucleic acid constructs of the invention are preferably integratedchromosomally.

In a preferred embodiment, the parent organisms used are organisms whichare already capable of producing the fine chemical of interest. Amongthe particularly preferred organisms of the bacteria of the genusCorynebacterium and the particularly preferred fine chemicals lysine,methionine and threonine, particular preference is given to those parentorganisms already capable of producing lysine. These are particularlypreferably corynebacteria in which, for example, the gene coding for anasparto-kinase (ask gene) is deregulated or feedback inhibition has beenremoved or reduced. For example, these kind of bacteria have a mutationin the ask gene which results in a reduction or removal of feedbackinhibition, such as, for example, the mutation T311I.

The invention therefore relates in particular to a genetically modifiedorganism obtainable by the above-described process.

The invention furthermore relates to a nonhuman, genetically modifiedorganism which has been transformed with

-   a) at least one above-described MP nucleic acid of the invention or-   b) at least one above-described nucleic acid construct of the    invention or-   c) a promoter which is heterologous with respect to the    above-described endogenous MP nucleic acid of the invention and    which enables said endogenous MP nucleic acid of the invention to be    expressed in said organism.

In another embodiment, an endogenous MP gene in the genome of the parentorganism has been modified, for example functionally disrupted, byhomologous recombination with a modified MP gene.

Preferably, expression of the nucleic acid of the invention results inthe modulation of production of a fine chemical from said organism incomparison with the parent organism.

Preferred nonhuman organisms are plants, algae and microorganisms.Preferred microorganisms are bacteria, yeasts or fungi. Particularlypreferred microorganisms are bacteria, in particular bacteria of thegenus Corynebacterium or Brevibacterium, with particular preferencebeing given to Corynebacterium glutamicum.

Particular preferred bacteria of the genus Corynebacterium orBrevibacterium as parent organisms or organisms or genetically modifiedorganisms are the bacteria listed in table 3 below.

TABLE 3 Bacterium Deposition number Genus species ATCC FERM NRRL CECTNCIMB CBS NCTC DSMZ Brevibacterium ammoniagenes 21054 Brevibacteriumammoniagenes 19350 Brevibacterium ammoniagenes 19351 Brevibacteriumammoniagenes 19352 Brevibacterium ammoniagenes 19353 Brevibacteriumammoniagenes 19354 Brevibacterium ammoniagenes 19355 Brevibacteriumammoniagenes 19356 Brevibacterium ammoniagenes 21055 Brevibacteriumammoniagenes 21077 Brevibacterium ammoniagenes 21553 Brevibacteriumammoniagenes 21580 Brevibacterium ammoniagenes 39101 Brevibacteriumbutanicum 21196 Brevibacterium divaricatum 21792 P928 Brevibacteriumflavum 21474 Brevibacterium flavum 21129 Brevibacterium flavum 21518Brevibacterium flavum B11474 Brevibacterium flavum B11472 Brevibacteriumflavum 21127 Brevibacterium flavum 21128 Brevibacterium flavum 21427Brevibacterium flavum 21475 Brevibacterium flavum 21517 Brevibacteriumflavum 21528 Brevibacterium flavum 21529 Brevibacterium flavum B11477Brevibacterium flavum B11478 Brevibacterium flavum 21127 Brevibacteriumflavum B11474 Brevibacterium healii 15527 Brevibacterium ketoglutamicum21004 Brevibacterium ketoglutamicum 21089 Brevibacterium ketosoreductum21914 Brevibacterium lactofermentum 70 Brevibacterium lactofermentum 74Brevibacterium lactofermentum 77 Brevibacterium lactofermentum 21798Brevibacterium lactofermentum 21799 Brevibacterium lactofermentum 21800Brevibacterium lactofermentum 21801 Brevibacterium lactofermentum B11470Brevibacterium lactofermentum B11471 Brevibacterium lactofermentum 21086Brevibacterium lactofermentum 21420 Brevibacterium lactofermentum 21086Brevibacterium lactofermentum 31269 Brevibacterium linens 9174Brevibacterium linens 19391 Brevibacterium linens 8377 Brevibacteriumparaffinolyticum 11160 Brevibacterium spec. 717.73 Brevibacterium spec.717.73 Brevibacterium spec. 14604 Brevibacterium spec. 21860Brevibacterium spec. 21864 Brevibacterium spec. 21865 Brevibacteriumspec. 21866 Brevibacterium spec. 19240 Corynebacterium acetoacidophilum21476 Corynebacterium acetoacidophilum 13870 Corynebacteriumacetoglutamicum B11473 Corynebacterium acetoglutamicum B11475Corynebacterium acetoglutamicum 15806 Corynebacterium acetoglutamicum21491 Corynebacterium acetoglutamicum 31270 Corynebacterium acetophilumB3671 Corynebacterium ammoniagenes 6872 2399 Corynebacteriumammoniagenes 15511 Corynebacterium fujiokense 21496 Corynebacteriumglutamicum 14067 Corynebacterium glutamicum 39137 Corynebacteriumglutamicum 21254 Corynebacterium glutamicum 21255 Corynebacteriumglutamicum 31830 Corynebacterium glutamicum 13032 Corynebacteriumglutamicum 14305 Corynebacterium glutamicum 15455 Corynebacteriumglutamicum 13058 Corynebacterium glutamicum 13059 Corynebacteriumglutamicum 13060 Corynebacterium glutamicum 21492 Corynebacteriumglutamicum 21513 Corynebacterium glutamicum 21526 Corynebacteriumglutamicum 21543 Corynebacterium glutamicum 13287 Corynebacteriumglutamicum 21851 Corynebacterium glutamicum 21253 Corynebacteriumglutamicum 21514 Corynebacterium glutamicum 21516 Corynebacteriumglutamicum 21299 Corynebacterium glutamicum 21300 Corynebacteriumglutamicum 39684 Corynebacterium glutamicum 21488 Corynebacteriumglutamicum 21649 Corynebacterium glutamicum 21650 Corynebacteriumglutamicum 19223 Corynebacterium glutamicum 13869 Corynebacteriumglutamicum 21157 Corynebacterium glutamicum 21158 Corynebacteriumglutamicum 21159 Corynebacterium glutamicum 21355 Corynebacteriumglutamicum 31808 Corynebacterium glutamicum 21674 Corynebacteriumglutamicum 21562 Corynebacterium glutamicum 21563 Corynebacteriumglutamicum 21564 Corynebacterium glutamicum 21565 Corynebacteriumglutamicum 21566 Corynebacterium glutamicum 21567 Corynebacteriumglutamicum 21568 Corynebacterium glutamicum 21569 Corynebacteriumglutamicum 21570 Corynebacterium glutamicum 21571 Corynebacteriumglutamicum 21572 Corynebacterium glutamicum 21573 Corynebacteriumglutamicum 21579 Corynebacterium glutamicum 19049 Corynebacteriumglutamicum 19050 Corynebacterium glutamicum 19051 Corynebacteriumglutamicum 19052 Corynebacterium glutamicum 19053 Corynebacteriumglutamicum 19054 Corynebacterium glutamicum 19055 Corynebacteriumglutamicum 19056 Corynebacterium glutamicum 19057 Corynebacteriumglutamicum 19058 Corynebacterium glutamicum 19059 Corynebacteriumglutamicum 19060 Corynebacterium glutamicum 19185 Corynebacteriumglutamicum 13286 Corynebacterium glutamicum 21515 Corynebacteriumglutamicum 21527 Corynebacterium glutamicum 21544 Corynebacteriumglutamicum 21492 Corynebacterium glutamicum B8183 Corynebacteriumglutamicum B8182 Corynebacterium glutamicum B12416 Corynebacteriumglutamicum B12417 Corynebacterium glutamicum B12418 Corynebacteriumglutamicum B11476 Corynebacterium glutamicum 21608 Corynebacteriumlilium P973 Corynebacterium nitrilophilus 21419 11594 Corynebacteriumspec. P4445 Corynebacterium spec. P4446 Corynebacterium spec. 31088Corynebacterium spec. 31089 Corynebacterium spec. 31090 Corynebacteriumspec. 31090 Corynebacterium spec. 31090 Corynebacterium spec. 1595420145 Corynebacterium spec. 21857 Corynebacterium spec. 21862Corynebacterium spec. 21863 The abbreviations have the followingmeaning: ATCC: American Type Culture Collection, Rockville, MD, USAFERM: Fermentation Research Institute, Chiba, Japan NRRL: ARS CultureCollection, Northern Regional Research Laboratory, Peoria, IL, USA CECT:Coleccion Espanola de Cultivos Tipo, Valencia, Spain NCIMB: NationalCollection of Industrial and Marine Bacteria Ltd., Aberdeen, UK CBS:Centraalbureau voor Schimmelcultures, Baarn, NL NCTC: NationalCollection of Type Cultures, London, UK DSMZ: Deutsche Sammlung vonMikroorganismen und Zellkulturen, Braunschweig, Germany

Another preferred embodiment are the genetically modified organisms ofthe invention, also referred to as “host cells” hereinbelow, which havemore than one of the MP nucleic acid molecules of the invention. Suchhost cells can be prepared in various ways known to the skilled worker.They may be transfected, for example, by vectors carrying several of thenucleic acid molecules of the invention. However, it is also possible touse a vector for introducing in each case one nucleic acid molecule ofthe invention into the host cell and therefore to use a variety ofvectors either simultaneously or sequentially. Thus it is possible toconstruct host cells which carry numerous, up to several hundred,nucleic acid sequences of the invention. Such an accumulation can oftenproduce superadditive effects on the host cell with respect to finechemical productivity.

In a preferred embodiment, the genetically modified organisms comprisein a chromosomally integrated manner at least two MP nucleic acids ofthe invention or a heterologous promoter functionally linked to anendogenous MP nucleic acid of the invention.

In another embodiment, the MP proteins and/or MP genes of the inventionare capable of modulating the production of a fine chemical of interestin an organism, in particular in corynebacteria or brevibacteria,particularly preferably in C. glutamicum. With the aid of generecombination techniques it is possible to manipulate one or moreinventive regulatory proteins for metabolic pathways such that theirfunction is modulated. For example, it is possible to improve abiosynthesis enzyme with respect to efficiency or to destroy itsallosteric control region so that feedback inhibition of the productionof the compound is prevented. Accordingly, a degradation enzyme may bedeleted or be modified by substitution, deletion or addition such thatits degradation activity for the compound of interest is reduced,without impairing cell viability. In any case, it is possible toincrease the overall yield or production rate of any of the said finechemicals of interest.

It is also possible that these modifications in the protein andnucleotide molecules of the invention can improve the production of finechemicals indirectly. The regulatory mechanisms of the metabolicpathways in the cell are inevitably linked and activation of onemetabolic pathway can cause repression or activation of anothermetabolic pathway in an accompanying manner. Modulating the activity ofone or more proteins of the invention can influence the production orthe efficiency of the activity of other fine-chemical biosynthetic ordegradation pathways. Reducing the ability of an MP protein to represstranscription of a gene which encodes a particular protein in amino acidbiosynthesis makes it possible to simultaneously derepress other aminoacid biosynthetic pathways, since these metabolic pathways are linked toone another. By modifying the MP proteins of the invention it ispossible to decouple to a certain degree cell growth and cell divisionfrom their extracellular environments; by influencing an MP proteinwhich usually represses the biosynthesis of a nucleotide when theextracellular conditions for growth and cell division are suboptimalsuch that it now lacks this function, it is possible to enable growth,even if the extracellular conditions are poor. This is of particularimportance for large-scale fermentative cultivation, for which theculture conditions with respect to temperature, nutrient supply oraeration are often suboptimal but still promote growth and cell divisionafter the cellular regulatory systems for such factors have beeneliminated.

The invention therefore furthermore relates to a process for preparing afine chemical by culturing an above-described, genetically modifiedorganism of the invention.

Furthermore, the invention relates to a process for preparing a finechemical by

A) transforming a nonhuman parent organism with

-   a) at least one above-described MP nucleic acid of the invention or-   b) at least one above-described nucleic acid construct of the    invention or-   c) a promoter which is heterologous with respect to the    above-described endogenous MP nucleic acid of the invention and    which enables said endogenous MP nucleic acid of the invention to be    expressed in said organism,    and    B) culturing the genetically modified organism prepared according to    feature A).

The genetically modified organism is cultured in a manner known per seand according to the organism. For example, the bacteria are cultured inliquid culture in suitable fermentation media.

In a preferred embodiment, at least one of the fine chemicals isisolated from the genetically modified organisms and/or the culturingmedium after the culturing step.

The term “fine chemical” is known in the art and includes compoundswhich are produced by an organism and are used in various branches ofindustry such as, for example, but not restricted to, the pharmaceuticalindustry, the agricultural industry, the cosmetics industry, the foodindustry and the feed industry. These compounds comprise organic acidssuch as, for example, tartaric acid, itaconic acid and diaminopimelicacid, both proteinogenic and nonproteinogenic amino acids, purine andpyrimidine bases, nucleosides and nucleotides (as described, forexample, in Kuninaka, A. (1996) Nucleotides and related compounds, pp.561-612, in Biotechnology Vol. 6, Rehm et al., editors VCH: Weinheim andthe references therein), lipids, saturated and unsaturated fatty acids(for example arachidonic acid), diols (e.g. propanediol and butanediol),carbohydrates (e.g. hyaluronic acid and trehalose), aromatic compounds(e.g. aromatic amines, vanillin and indigo), vitamins and cofactors (asdescribed in Ullmann's Encyclopedia of Industrial Chemistry, Vol. A27,“Vitamins”, pp. 443-613 (1996) VCH: Weinheim and the references therein;and Ong, A. S., Niki, E. and Packer, L. (1995) “Nutrition, Lipids,Health and Disease” Proceedings of the UNESCO/Confederation ofScientific and Technological Associations in Malaysia and the Societyfor Free Radical Research—Asia, held Sep. 1-3, 1994, in Penang,Malaysia, AOCS Press (1995)), enzymes and all other chemicals describedby Gutcho (1983) in Chemicals by Fermentation, Noyes Data Corporation,ISBN: 0818805086 and the references indicated therein. The metabolismand the uses of particular fine chemicals are further illustrated below.

I. Amino Acid Metabolism and Uses

Amino acids comprise the fundamental structural units of all proteinsand are thus essential for normal functions of the cell. The term “aminoacid” is known in the art. Proteinogenic amino acids, of which there are20 types, serve as structural units for proteins, in which they arelinked together by peptide bonds, whereas the nonproteinogenic aminoacids (hundreds of which are known) usually do not occur in proteins(see Ullmann's Encyclopedia of Industrial Chemistry, Vol. A2, pp. 57-97VCH: Weinheim (1985)). Amino acids can exist in the D or Lconfiguration, although L-amino acids are usually the only type found innaturally occurring proteins. Biosynthetic and degradation pathways ofeach of the 20 proteinogenic amino acids are well characterized both inprokaryotic and eukaryotic cells (see, for example, Stryer, L.Biochemistry, 3rd edition, pp. 578-590 (1988)). The “essential” aminoacids (histidine, isoleucine, leucine, lysine, methionine,phenylalanine, threonine, tryptophan and valine), so called because,owing to the complexity of their biosynthesis, they must be taken inwith the diet, are converted by simple biosynthetic pathways into theother 11 “nonessential” amino acids (alanine, arginine, asparagine,aspartate, cysteine, glutamate, glutamine, glycine, proline, serine andtyrosine). Higher animals are able to synthesize some of these aminoacids but the essential amino acids must be taken in with the food inorder that normal protein synthesis takes place.

Apart from their function in protein biosynthesis, these amino acids areinteresting chemicals as such, and it has been found that many havevarious applications in the food, animal feed, chemicals, cosmetics,agricultural and pharmaceutical industries. Lysine is an important aminoacid not only for human nutrition but also for monogastric livestocksuch as poultry and pigs. Glutamate is most frequently used as flavoradditive (monosodium glutamate, MSG) and elsewhere in the food industry,as are aspartate, phenylalanine, glycine and cysteine. Glycine,L-methionine and tryptophan are all used in the pharmaceutical industry.Glutamine, valine, leucine, isoleucine, histidine, arginine, proline,serine and alanine are used in the pharmaceutical industry and thecosmetics industry. Threonine, tryptophan and D/L-methionine are widelyused animal feed additives (Leuchtenberger, W. (1996) Aminoacids—technical production and use, pp. 466-502 in Rehm et al.,(editors) Biotechnology Vol. 6, Chapter 14a, VCH: Weinheim). It has beenfound that these amino acids are additionally suitable as precursors forsynthesizing synthetic amino acids and proteins, such asN-acetylcysteine, S-carboxymethyl-L-cysteine, (S)-5-hydroxytryptophanand other substances described in Ullmann's Encyclopedia of IndustrialChemistry, Vol. A2, pp. 57-97, VCH, Weinheim, 1985.

The biosynthesis of these natural amino acids in organisms able toproduce them, for example bacteria, has been well characterized (for areview of bacterial amino acid biosynthesis and its regulation, seeUmbarger, H. E. (1978) Ann. Rev. Biochem. 47: 533-606). Glutamate issynthesized by reductive amination of α-ketoglutarate, an intermediateproduct in the citric acid cycle. Glutamine, proline and arginine areeach generated successively from glutamate. The biosynthesis of serinetakes place in a three-step process and starts with 3-phosphoglycerate(an intermediate product of glycolysis), and affords this amino acidafter oxidation, transamination and hydrolysis steps. Cysteine andglycine are each produced from serine, specifically the former bycondensation of homocysteine with serine, and the latter by transfer ofthe side-chain β-carbon atom to tetrahydrofolate in a reaction catalyzedby serine transhydroxymethylase. Phenylalanine and tyrosine aresynthesized from the precursors of the glycolysis and pentose phosphatepathway, erythrose 4-phosphate and phosphoenolpyruvate in a 9-stepbiosynthetic pathway which diverges only in the last two steps after thesynthesis of prephenate. Tryptophan is likewise produced from these twostarting molecules but it is synthesized by an 11-step pathway. Tyrosinecan also be prepared from phenylalanine in a reaction catalyzed byphenylalanine hydroxylase. Alanine, valine and leucine are eachbiosynthetic products derived from pyruvate, the final product ofglycolysis. Aspartate is formed from oxalacetate, an intermediateproduct of the citrate cycle. Asparagine, methionine, threonine andlysine are each produced by the conversion of aspartate. Isoleucine isformed from threonine. Histidine is formed from 5-phosphoribosyl1-pyrophosphate, an activated sugar, in a complex 9-step pathway.

Amounts of amino acids exceeding those required for protein biosynthesisby the cell cannot be stored and are instead broken down, so thatintermediate products are provided for the principal metabolic pathwaysin the cell (for a review, see Stryer, L., Biochemistry, 3rd edition,Chapter 21 “Amino Acid Degradation and the Urea Cycle”; pp. 495-516(1988)). Although the cell is able to convert unwanted amino acids intothe useful intermediate products of metabolism, production of aminoacids is costly in terms of energy, the precursor molecules and theenzymes necessary for their synthesis. It is therefore not surprisingthat amino acid biosynthesis is regulated by feedback inhibition,whereby the presence of a particular amino acid slows down or completelystops its own production (for a review of the feedback mechanism inamino acid biosynthetic pathways, see Stryer, L., Biochemistry, 3rdedition, Chapter 24, “Biosynthesis of Amino Acids and Heme”, pp. 575-600(1988)). The output of a particular amino acid is therefore restrictedby the amount of this amino acid in the cell.

II. Vitamins, Cofactors and Nutraceutical Metabolism and Uses

Vitamins, cofactors and nutraceuticals comprise another group ofmolecules. Higher animals have lost the ability to synthesize them andtherefore have to take them in, although they are easily synthesized byother organisms such as bacteria. These molecules are either bioactivemolecules per se or precursors of bioactive substances which serve aselectron carriers or intermediate products in a number of metabolicpathways. Besides their nutritional value, these compounds also have asignificant industrial value as colorants, antioxidants and catalysts orother processing auxiliaries. (For a review of the structure, activityand industrial applications of these compounds, see, for example,Ullmann's Encyclopedia of Industrial Chemistry, “Vitamins”, Vol. A27,pp. 443-613, VCH: Weinheim, 1996.) The term “vitamin” is known in theart and comprises nutrients which are required for normal functioning ofan organism but cannot be synthesized by this organism itself. The groupof vitamins may include cofactors and nutraceutical compounds. The term“cofactor” comprises nonproteinaceous compounds necessary for theappearance of normal enzymic activity. These compounds may be organic orinorganic; the cofactor molecules of the invention are preferablyorganic. The term “nutraceutical” comprises food additives which arehealth-promoting in plants and animals, especially humans. Examples ofsuch molecules are vitamins, antioxidants and likewise certain lipids(e.g. polyunsaturated fatty acids).

The biosynthesis of these molecules in organisms able to produce them,such as bacteria, has been comprehensively characterized (Ullmann'sEncyclopedia of Industrial Chemistry, “Vitamins”, Vol. A27, pp. 443-613,VCH: Weinheim, 1996, Michal, G. (1999) Biochemical Pathways: An Atlas ofBiochemistry and Molecular Biology, John Wiley & Sons; Ong, A. S., Niki,E. and Packer, L. (1995) “Nutrition, Lipids, Health and Disease”Proceedings of the UNESCO/Confederation of Scientific and TechnologicalAssociations in Malaysia and the Society for Free Radical Research—Asia,held on Sep. 1-3, 1994, in Penang, Malaysia, AOCS Press, Champaign, Ill.X, 374 S).

Thiamine (vitamin B₁) is formed by chemical coupling of pyrimidine andthiazole units. Riboflavin (vitamin B₂) is synthesized from guanosine5′-triphosphate (GTP) and ribose 5′-phosphate. Riboflavin in turn isemployed for the synthesis of flavin mononucleotide (FMN) and flavinadenine dinucleotide (FAD). The family of compounds together referred toas “vitamin B6” (for example pyridoxine, pyridoxamine, pyridoxal5′-phosphate and the commercially used pyridoxine hydrochloride) are allderivatives of the common structural unit 5-hydroxy-6-methylpyridine.Pantothenate (pantothenic acid,R-(+)-N-(2,4-dihydroxy-3,3-dimethyl-1-oxobutyl)-β-alanine) can beprepared either by chemical synthesis or by fermentation. The last stepsin pantothenate biosynthesis consist of ATP-driven condensation ofβ-alanine and pantoic acid. The enzymes responsible for the biosyntheticsteps for the conversion into pantoic acid and into β-alanine and forthe condensation to pantothenic acid are known. The metabolically activeform of pantothenate is coenzyme A, whose biosynthesis takes place by 5enzymatic steps. Pantothenate, pyridoxal 5′-phosphate, cysteine and ATPare the precursors of coenzyme A. These enzymes catalyze not only theformation of pantothenate but also the production of (R)-pantoic acid,(R)-pantolactone, (R)-panthenol (provitamin B₅), pantetheine (and itsderivatives) and coenzyme A.

The biosynthesis of biotin from the precursor molecule pimeloyl-CoA inmicroorganisms has been investigated in detail, and several of the genesinvolved have been identified. It has emerged that many of thecorresponding proteins are involved in the Fe cluster synthesis andbelong to the class of nifS proteins. Liponic acid is derived fromoctanoic acid and serves as coenzyme in energy metabolism, where it is aconstituent of the pyruvate dehydrogenase complex and of theα-ketoglutarate dehydrogenase complex. Folates are a group of substancesall derived from folic acid, which in turn is derived from L-glutamicacid, p-aminobenzoic acid and 6-methylpterin. The biosynthesis of folicacid and its derivatives starting from the metabolic intermediateproducts guanosine 5′-triphosphate (GTP), L-glutamic acid andp-aminobenzoic acid has been investigated in detail in certainmicroorganisms.

Corrinoids (such as the cobalamines and, in particular, vitamin B₁₂) andthe porphyrins belong to a group of chemicals distinguished by atetrapyrrole ring system. The biosynthesis of vitamin B₁₂ is so complexthat it has not yet been completely characterized, but many of theenzymes and substrates involved are now known. Nicotinic acid(nicotinate) and nicotinamide are pyridine derivatives which are alsoreferred to as “niacin”. Niacin is the precursor of the importantcoenzymes NAD (nicotinamide adenine dinucleotide) and NADP (nicotinamideadenine dinucleotide phosphate) and their reduced forms.

Production of these compounds on the industrial scale is mostly based oncell-free chemical syntheses, although some of these chemicals havelikewise been produced by large-scale cultivation of microorganisms,such as riboflavin, vitamin B₆, pantothenate and biotin. Only vitaminB₁₂ is, because of the complexity of its synthesis, produced only byfermentation. In vitro processes require a considerable expenditure ofmaterials and time and frequently high costs.

III. Purine, Pyrimidine, Nucleoside and Nucleotide Metabolism and Uses

Genes for purine and pyrimidine metabolism and their correspondingproteins are important aims for the therapy of oncoses and viralinfections. The term “purine” or “pyrimidine” comprisesnitrogen-containing bases which form part of nucleic acids, coenzymesand nucleotides. The term “nucleotide” encompasses the fundamentalstructural units of nucleic acid molecules, which comprise anitrogen-containing base, a pentose sugar (the sugar is ribose in thecase of RNA and the sugar is D-deoxyribose in the case of DNA) andphosphoric acid. The term “nucleoside” comprises molecules which serveas precursors of nucleotides but have, in contrast to the nucleotides,no phosphoric acid unit. It is possible to inhibit RNA and DNA synthesisby inhibiting the biosynthesis of these molecules or their mobilizationto form nucleic acid molecules; targeted inhibition of this activity incancerous cells allows the ability of tumor cells to divide andreplicate to be inhibited.

There are also nucleotides which do not form nucleic acid molecules butserve as energy stores (i.e. AMP) or as coenzymes (i.e. FAD and NAD).

Several publications have described the use of these chemicals for thesemedical indications, the purine and/or pyrimidine metabolism beinginfluenced (for example Christopherson, R. I. and Lyons, S. D. (1990)“Potent inhibitors of de novo pyrimidine and purine biosynthesis aschemotherapeutic agents”, Med. Res. Reviews 10: 505-548). Investigationsof enzymes involved in purine and pyrimidine metabolism haveconcentrated on the development of novel medicaments which can be used,for example, as immunosuppressants or antiproliferative agents (Smith,J. L. “Enzymes in Nucleotide Synthesis” Curr. Opin. Struct. Biol. 5(1995) 752-757; Simmonds, H. A., Biochem. Soc. Transact. 23 (1995)877-902). However, purine and pyrimidine bases, nucleosides andnucleotides also have other possible uses: as intermediate products inthe biosynthesis of various fine chemicals (e.g. thiamine,S-adenosylmethionine, folates or riboflavin), as energy carriers for thecell (for example ATP or GTP) and for chemicals themselves, areordinarily used as flavor enhancers (for example IMP or GMP) or for manymedical applications (see, for example, Kuninaka, A., (1996)“Nucleotides and Related Compounds in Biotechnology” Vol. 6, Rehm etal., editors VCH: Weinheim, pp. 561-612). Enzymes involved in purine,pyrimidine, nucleoside or nucleotide metabolism are also increasinglyserving as targets against which chemicals are being developed for cropprotection, including fungicides, herbicides and insecticides.

The metabolism of these compounds in bacteria has been characterized(for reviews, see, for example, Zalkin, H. and Dixon, J. E. (1992) “Denovo purine nucleotide biosynthesis” in Progress in Nucleic AcidsResearch and Molecular biology, Vol. 42, Academic Press, pp. 259-287;and Michal, G. (1999) “Nucleotides and Nucleosides”; Chapter 8 in:Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology,Wiley, New York). Purine metabolism, the object of intensive research,is essential for normal functioning of the cell. Disordered purinemetabolism in higher animals may cause severe illnesses, for examplegout. Purine nucleotides are synthesized from ribose 5-phosphate by anumber of steps via the intermediate compound inosine 5′-phosphate(IMP), leading to the production of guanosine 5′-monophosphate (GMP) oradenosine 5′-monophosphate (AMP), from which the triphosphate forms usedas nucleotides can easily be prepared. These compounds are also used asenergy stores, so that breakdown thereof provides energy for manydifferent biochemical processes in the cell. Pyrimidine biosynthesistakes place via formation of uridine 5′-monophosphate (UMP) from ribose5-phosphate. UMP in turn is converted into cytidine 5′-triphosphate(CTP). The deoxy forms of all nucleotides are prepared in a one-stepreduction reaction from the diphosphate ribose form of the nucleotide togive the diphosphate deoxyribose form of the nucleotide. Afterphosphorylation, these molecules can take part in DNA synthesis.

IV. Trehalose Metabolism and Uses

Trehalose consists of two glucose molecules linked together by α,α-1,1linkage. It is ordinarily used in the food industry as sweetener, asadditive for dried or frozen foods and in beverages. However, it is alsoused in the pharmaceutical industry or in the cosmetics industry andbiotechnology industry (see, for example, Nishimoto et al., (1998) U.S.Pat. No. 5,759,610; Singer, M. A. and Lindquist, S. Trends Biotech. 16(1998) 460-467; Paiva, C. L. A. and Panek, A. D. Biotech Ann. Rev. 2(1996) 293-314; and Shiosaka, M. J. Japan 172 (1997) 97-102). Trehaloseis produced by enzymes of many microorganisms and is naturally releasedinto the surrounding medium, from which it can be isolated by methodsknown in the art.

Particularly preferred fine chemicals are amino acids, in particularamino acids selected from the group consisting of L-lysine, L-threonineand L-methionine.

Another aspect of the invention relates to processes for modulating theproduction of a fine chemical from a nonhuman organism. These processescomprise contacting the cell with a substance which modulates the MPprotein activity or MP nucleic acid expression such that acell-associated activity is modified in comparison with the sameactivity in the absence of said substance. In a preferred embodiment,the cell is modulated with respect to one or more regulatory systems formetabolic pathways in organisms, in particular in bacteria of the genusCorynebacterium and/or Brevibacterium, in particular C. glutamicum, sothat this host cell gives improved yields or an improved production rateof a fine chemical of interest. The substance which modulates the MPprotein activity stimulate, for example, MP protein activity or MPnucleic acid expression. Examples of substances stimulating MP proteinactivity or MP nucleic acid expression include small molecules, activeMP proteins and nucleic acids which encode MP proteins and have beenintroduced into the cell. Examples of substances which inhibit MPactivity or MP expression include small molecules and antisense MPnucleic acid molecules.

Another aspect of the invention relates to processes for modulating theyields of a compound of interest from a cell, comprising introducing anMP gene into a cell, which gene either remains on a separate plasmid oris integrated into the genome of the host cell. Integration into thegenome may take place randomly or via homologous recombination such thatthe native gene is replaced by the integrated copy, leading to theproduction of the compound of interest from the cell to be modulated. Ina preferred embodiment, these yields are increased.

In another preferred embodiment, the fine chemical is an amino acid. Ina particularly preferred embodiment, this amino acid is L-lysine,L-methionine or L-threonine.

The following subsections describe various aspects and preferredembodiments of the invention in more detail:

A. Isolated Nucleic Acid Molecules

The term “nucleic acid molecule”, as used herein, is intended tocomprise DNA molecules (e.g. cDNA or genomic DNA) and RNA molecules(e.g. mRNA) and also DNA or RNA analogs which are generated by means ofnucleotide analogs. Moreover, this term comprises the untranslatedsequence located at the 3′ and 5′ end of the coding gene region: atleast about 100 nucleotides of the sequence upstream of the 5′ end ofthe coding region and at least about 20 nucleotides of the sequencedownstream of the 3′ end of the coding gene region.

The nucleic acid molecule may be single-stranded or double-stranded butis preferably a double-stranded DNA. An “isolated” nucleic acid moleculeis removed from other nucleic acid molecules which are present in thenatural source of the nucleic acid. An “isolated” nucleic acidpreferably does not have any sequences which flank the nucleic acidnaturally in the genomic DNA of the organism from which the nucleic acidoriginates (for example sequences located at the 5′ or 3′ end of thenucleic acid).

In various embodiments, the isolated MP nucleic acid molecule may have,for example, less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1kb of the nucleotide sequences which naturally flank the nucleic acidmolecule in the genomic DNA of the cell from which the nucleic acidoriginates (e.g. a C. glutamicum cell). In addition to this, an“isolated” nucleic acid molecule, such as a cDNA molecule, may beessentially free of another cellular material or culture medium, if itis prepared by recombinant techniques, or free of chemical precursors orother chemicals, if it is chemically synthesized.

Moreover, a nucleic acid molecule can be isolated via polymerase chainreaction, using the oligonucleotide primer produced on the basis of thissequence (for example, it is possible to isolate a nucleic acid moleculecomprising a complete sequence from Appendix A or a section thereof viapolymerase chain reaction by using oligonucleotide primers which havebeen produced on the basis of this same sequence from Appendix A). Forexample, mRNA can be isolated from normal endothelial cells (for examplevia the guanidinium thiocyanate extraction method of Chirgwin et al.(1979) Biochemistry 18: 5294-5299), and the cDNA can be prepared bymeans of reverse transcriptase (e.g. Moloney-MLV reverse transcriptase,available from Gibco/BRL, Bethesda, Md., or AMV reverse transcriptase,available from Seikagaku America, Inc., St. Petersburg, Fla.). Syntheticoligonucleotide primers for amplification via polymerase chain reactioncan be produced on the basis of any of the nucleotide sequences shown inAppendix A. A nucleic acid of the invention may be amplified by means ofcDNA or, alternatively, genomic DNA as template and suitableoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid amplified in this way may be cloned into asuitable vector and characterized by DNA sequence analysis.Oligonucleotides corresponding to an MP nucleotide sequence may beprepared by standard syntheses using, for example, an automatic DNAsynthesizer.

In a preferred embodiment, an isolated nucleic acid molecule of theinvention comprises any of the nucleotide sequences listed in table1/column 1 containing a back-translated mutation corresponding to theamino acid position according to table 1/column 4.

In a further preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule complementary to any ofthe above-described nucleotide sequences, or a section thereof, saidnucleic acid molecule being sufficiently complementary to any of theabove-described nucleotide sequences for it to be able to hybridize withany of the sequences described above, resulting in a stable duplex.

Sections of proteins encoded by the MP nucleic acid molecules of theinvention are preferably biologically active sections of any of the MPproteins. The term “biologically active section of an MP protein”, asused herein, is intended to comprise a section, for example a domain ora motif, of an MP protein, which can transcriptionally, translationallyor posttranslationally regulate a metabolic pathway in C. glutamicum orhas an activity indicated in table 1. In order to determine whether anMP protein or a biologically active section thereof can regulate ametabolic pathway in C. glutamicum transcriptionally, translationally orposttranslationally, an enzyme activity assay may be carried out. Theseassay methods, as described in detail in example 8 of the Examplessection, are familiar to the skilled worker.

In addition to further naturally occurring MP sequence variants whichmay exist in the population, the skilled worker also understands thatfurther changes can be introduced into a nucleotide sequence of table 1via further mutation, leading to a further change in the amino acidsequence of the encoded MP protein in comparison with the wild type,without impairing the functionality of the MP protein. Thus is itpossible, for example, to prepare in a sequence of table 1 nucleotidesubstitutions which lead to amino acid substitutions at “nonessential”amino acid residues. A “nonessential” amino acid residue in a wild-typesequence of any of the MP proteins (table 1) can be modified withoutmodifying the activity of said MP protein, whereas an “essential” aminoacid residue is required for MP protein activity. However, other aminoacid residues (for example non-conserved or merely semiconserved aminoacid residues in the domain having MP activity) may be nonessential foractivity and can therefore probably be modified without modifying the MPactivity.

B. Recombinant Expression Vectors and Host Cells

Another aspect of the invention relates to nucleic acid constructs suchas, for example, vectors, preferably expression vectors, containing aninventive nucleic acid which encodes an MP protein. The term “vector”,as used herein, relates to a nucleic acid molecule capable oftransporting another nucleic acid to which it is bound.

One type of vector is a “plasmid”, which term means a circulardouble-stranded DNA loop into which additional DNA segments can beligated. Another type of vector is a viral vector, allowing additionalDNA segments to be ligated into the viral genome. Some vectors arecapable of replicating autonomously in a host cell into which they havebeen introduced (for example bacterial vectors with bacterial origin ofreplication and episomal mammalian vectors).

Other vectors (e.g. nonepisomal mammalian vectors) are integrated intothe genome of a host cell when introduced into said host cell andthereby replicated together with the host genome.

Moreover, particular vectors are capable of controlling the expressionof genes to which they are functionally linked. These vectors arereferred to as “expression vectors”. Normally, expression vectors usedin DNA recombination techniques are in the form of plasmids. In thepresent specification, “plasmid” and “vector” may be usedinterchangeably, since the plasmid is the most commonly used type ofvector. The invention is intended to comprise said other types ofexpression vectors, such as viral vectors (for examplereplication-deficient retroviruses, adenoviruses and adeno-relatedviruses), which exert similar functions.

The recombinant expression vector of the invention comprises a nucleicacid of the invention in a form which is suitable for expressing saidnucleic acid in a host cell, meaning that the recombinant expressionvectors comprise one or more regulatory sequences which are selected onthe basis of the host cells to be used for expression and which arefunctionally linked to the nucleic acid sequence to be expressed. In arecombinant expression vector, the term “functionally linked” means thatthe nucleotide sequence of interest is bound to the regulatorysequence(s) such that expression of said nucleotide sequence is possible(for example in an in vitro transcription/translation system or in ahost cell, if the vector has been introduced into said host cell). Theterm “regulatory sequence” is intended to comprise promoters, enhancersand other expression control elements (e.g. polyadenylation signals).These regulatory sequences are described, for example, in Goeddel: GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). Regulatory sequences comprise those which controlconstitutive expression of a nucleotide sequence in many types of hostcells and those which control direct expression of said nucleotidesequence only in particular host cells. The skilled worker understandsthat designing an expression vector may depend on factors such as thechoice of host cell to be transformed, the extent of expression of theprotein of interest, etc. The expression vectors of the invention may beintroduced into the host cells in order to prepare proteins or peptides,including fusion proteins or fusion peptides, which are encoded by thenucleic acids as described herein (e.g. MP proteins, mutated forms of MPproteins, fusion proteins, etc.).

The recombinant expression vectors of the invention may be designed forexpressing MP proteins in prokaryotic or eukaryotic cells. For example,MP genes may be expressed in bacterial cells such as C. glutamicum,insect cells (using Baculovirus expression vectors), yeast cells andother fungal cells (see Romanos, M. A. et al. (1992) “Foreign geneexpression in yeast: a review”, Yeast 8: 423-488; van den Hondel, C. A.M. J. J. et al. (1991) “Heterologous gene expression in filamentousfungi” in: More Gene Manipulations in Fungi, J. W. Bennet & L. L.Lasure, editors, pp. 396-428: Academic Press: San Diego; and van denHondel, C. A. M. J. J. & Punt, P. J. (1991) “Gene transfer systems andvector development for filamentous fungi” in: Applied Molecular Geneticsof Fungi, Peberdy, J. F. et al., editors, pp. 1-28, Cambridge UniversityPress: Cambridge), algal and multicellular plant cells (see Schmidt, R.and Willmitzer, L. (1988) “High efficiency Agrobacteriumtumefaciens-mediated transformation of Arabidopsis thaliana leaf andcotyledon explants” Plant Cell Rep.: 583-586) or mammalian cells.Suitable host cells are further discussed in Goeddel, Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990). Alternatively, the recombinant expression vector may betranscribed and translated in vitro, for example by using T7 promoterregulatory sequences and T7 polymerase.

Proteins are expressed in prokaryotes mainly by using vectors containingconstitutive or inducible promoters which control the expression offusion or nonfusion proteins. Fusion vectors contribute a number ofamino acids to a protein encoded therein, usually at the amino terminusof the recombinant protein. These fusion vectors usually have threetasks: 1) enhancing the expression of recombinant protein; 2) increasingthe solubility of the recombinant protein; and 3) supporting thepurification of the recombinant protein by acting as a ligand inaffinity purification. Often a proteolytic cleavage site is introducedinto fusion expression vectors at the junction of fusion unit andrecombinant protein so that the recombinant protein can be separatedfrom the fusion unit after purifying the fusion protein. These enzymesand their corresponding recognition sequences comprise factor Xa,thrombin and enterokinase.

Common fusion expression vectors comprise pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. (1988) Gene 67: 31-40), pMAL (NewEngland Biolabs, Beverly, Mass.) and pRIT 5 (Pharmacia, Piscataway,N.J.), in which glutathione S-transferase (GST), maltose E-bindingprotein and protein A, respectively, are fused to the recombinant targetprotein. In one embodiment, the coding sequence of the MP protein iscloned into a pGEX expression vector such that a vector is generatedwhich encodes a fusion protein comprising, from N terminus to Cterminus, GST—thrombin cleavage site—protein X. The fusion protein maybe purified via affinity chromatography by means of aglutathione-agarose resin. The recombinant MP protein which is not fusedto GST may be obtained by cleaving the fusion protein with thrombin.

Examples of suitable inducible nonfusion E. coli expression vectorsinclude pTrc (Amann et al., (1988) Gene 69: 301-315) and pET 11d(Studier et al. Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. (1990) 60-89). The target geneexpression from the pTrc vector is based on transcription from a hybridtrp-lac fusion promoter by host RNA polymerase. The target geneexpression from the pET11d vector is based on transcription from aT7-gn10-lac fusion promoter, which is mediated by a coexpressed viralRNA polymerase (T7 gn1). This viral polymerase is provided in the BL 21(DE3) or HMS174 (DE3) host strain by a resident λ prophage which harborsa T7 gn1 gene under the transcriptional control of the lacUV 5 promoter.

One strategy for maximizing expression of the recombinant protein is toexpress said protein in a host bacterium whose ability toproteolytically cleave said recombinant protein is disrupted (Gottesman,S. Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 119-128). Another strategy is to modifythe nucleic acid sequence of the nucleic acid to be inserted into anexpression vector such that the individual codons for each amino acidare those which are preferably used in a bacterium selected forexpression, such as C. glutamicum (Wada et al. (1992) Nucleic Acids Res.20: 2111-2118). This modification of the nucleic acid sequences of theinvention is carried out by standard techniques of DNA synthesis.

In a further embodiment, the MP protein expression vector is anexpression vector of yeast. Examples of vectors for expression in theyeast S. cerevisiae include pYepSec1 (Baldari et al., (1987) Embo J. 6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30: 933-943), pJRY88(Schultz et al. (1987) Gene 54: 113-123) and pYES2 (InvitrogenCorporation, San Diego, Calif.). Vectors and methods for constructingvectors which are suitable for use in other fungi such as filamentousfungi include those which are described in detail in: van den Hondel, C.A. M. J. J. & Punt, P. J. (1991) “Gene transfer systems and vectordevelopment for filamentous fungi”, in: Applied Molecular Genetics offungi, J. F. Peberdy et al., editors, pp. 1-28, Cambridge UniversityPress: Cambridge.

As an alternative, it is possible to express the MP proteins of theinvention in insect cells using baculovirus expression vectors.Baculovirus vectors available for the expression of proteins in culturedinsect cells (e.g. Sf9 cells) include the pAc series (Smith et al.,(1983) Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow andSummers (1989) Virology 170: 31-39).

In a further embodiment, the MP proteins of the invention may beexpressed in unicellular plant cells (such as algae) or in cells ofhigher plants (e.g. spermatophytes such as crops). Examples ofexpression vectors of plants include those which are described in detailin Becker, D., Kemper, E., Schell, J. and Masterson, R. (1992) “Newplant binary vectors with selectable markers located proximal to theleft border”, Plant Mol. Biol. 20: 1195-1197; and Bevan, M. W. (1984)“Binary Agrobacterium vectors for plant transformation”, Nucl. AcidsRes. 12: 8711-8721.

In another embodiment, a nucleic acid of the invention is expressed inmammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, B. (1987) Nature329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6: 187-195). Whenused in mammalian cells, the control functions of the expression vectorare often provided by viral regulatory elements. Commonly used promotersare derived, for example, from polyoma, adenovirus2, cytomegalovirus andsimian virus 40. Other suitable expression systems for prokaryotic andeukaryotic cells can be found in Chapters 16 and 17 of Sambrook, J.,Fritsch, E. F. and Maniatis, T., Molecular cloning: A Laboratory Manual,2nd edition, Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989.

In a further embodiment, the recombinant mammalian expression vector maycause expression of the nucleic acid, preferably in a particular celltype (for example, tissue-specific regulatory elements are used forexpressing the nucleic acid). Tissue-specific regulatory elements areknown in the art. Nonlimiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv. Immunol. 43: 235-275), in particular promoters ofT-cell receptors (Winoto and Baltimore (1989) EMBO J. 8: 729-733) andimmunoglobulins (Banerji et al. (1983) Cell 33: 729-740; Queen andBaltimore (1983) Cell 33: 741-748), neuron-specific promoters (e.g.neurofilament promoter; Byrne and Ruddle (1989) PNAS 86: 5473-5477),pancreas-specific promoters (Edlund et al., (1985) Science 230: 912-916)and mamma-specific promoters (e.g. milk serum promoter; U.S. Pat. No.4,873,316 and European Patent Application document No. 264 166).Development-regulated promoters, for example the murine hox promoters(Kessel and Gruss (1990) Science 249: 374-379) and the α-fetoproteinpromoter (Campes and Tilghman (1989) Genes Dev. 3: 537-546), arelikewise included.

In addition, the invention provides a recombinant expression vectorcomprising an inventive DNA molecule which has been cloned into theexpression vector in antisense direction. This means that the DNAmolecule is functionally linked to a regulatory sequence such that anRNA molecule which is antisense to the MP mRNA can be expressed (viatranscription of the DNA molecule). It is possible to select regulatorysequences which are functionally bound to a nucleic acid cloned inantisense direction and which control the continuous expression of theantisense RNA molecule in a multiplicity of cell types; for example, itis possible to select viral promoters and/or enhancers or regulatorysequences which control the constitutive tissue-specific or celltype-specific expression of antisense RNA. The antisense expressionvector may be in the form of a recombinant plasmid, phagemid orattenuated virus, which produces antisense nucleic acids under thecontrol of a highly effective regulatory region whose activity isdetermined by the cell type into which the vector is introduced. Theregulation of gene expression by means of antisense genes is discussedin Weintraub, H. et al., Antisense RNA as a molecular tool for geneticanalysis, Reviews—Trends in Genetics, Vol. 1(1) 1986.

Another aspect of the invention relates to the host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. Naturally, these terms relate not only to a particular targetcell but also to the progeny or potential progeny of this cell. Sinceparticular modifications may appear in successive generations, due tomutation or environmental factors, this progeny is not necessarilyidentical with the parental cell but is still included in the scope ofthe term as used herein.

A host cell may be a prokaryotic or eukaryotic cell. For example, an MPprotein may be expressed in bacterial cells such as C. glutamicum,insect cells, yeast or mammalian cells (such as Chinese hamster ovary(CHO) cells or COS cells). Other suitable host cells are familiar to theskilled worker. Microorganisms which are related to Corynebacteriumglutamicum and can be used in a suitable manner as host cells for thenucleic acid and protein molecules of the invention are listed in table3.

Conventional transformation or transfection methods can be used tointroduce vector DNA into prokaryotic or eukaryotic cells. The terms“transformation” and “transfection”, as used herein, are intended tocomprise a multiplicity of methods known in the art for introducingforeign nucleic acid (e.g. DNA) into a host cell, including calciumphosphate or calcium chloride coprecipitation, DEAE-dextran-mediatedtransfection, lipofection or electroporation. Suitable methods fortransformation or transfection of host cells can be found in Sambrook etal. (Molecular Cloning: A Laboratory Manual. 2nd edition Cold SpringHarbor Laboratory, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989) and other laboratory manuals.

In the case of stable transfection of mammalian cells, it is known that,depending on the expression vector used and transfection technique used,only a small proportion of the cells integrate the foreign DNA intotheir genome. These integrants are usually identified and selected byintroducing a gene which encodes a selectable marker (e.g. resistance toantibiotics) together with the gene of interest into the host cells.Preferred selectable markers include those which impart resistance todrugs such as G418, hygromycin and methotrexate. A nucleic acid whichencodes a selectable marker may be introduced into a host cell on thesame vector that encodes an MP protein or may be introduced on aseparate vector. Cells which have been stably transfected with theintroduced nucleic acid may be identified by drug selection (forexample, cells which have integrated the selectable marker survive,whereas the other cells die).

A homologously recombined microorganism is generated by preparing avector which contains at least one MP gene section into which adeletion, addition or substitution has been introduced in order tomodify or functionally disrupt the MP gene. Said MP gene is preferably aCorynebacterium glutamicum MP gene, but it is also possible to use ahomolog from a related bacterium or even from a mammalian, yeast orinsect source. In a preferred embodiment, the vector is designed suchthat homologous recombination functionally disrupts the endogenous MPgene (i.e. the gene no longer encodes a functional protein; likewisereferred to as “knockout” vector). As an alternative, the vector may bedesigned such that homologous recombination mutates or otherwisemodifies the endogenous MP gene which, however, still encodes thefunctional protein (for example, the regulatory region located upstreammay be modified such that thereby the expression of the endogenous MPprotein is modified.). The modified MP gene section in the homologousrecombination vector is flanked at its 5′ and 3′ ends by additionalnucleic acid of the MP gene, which makes possible a homologousrecombination between the exogenous MP gene carried by the vector and anendogenous MP gene in a microorganism. The length of the additionalflanking MP nucleic acid is sufficient for a successful homologousrecombination with the endogenous gene. Usually, the vector containsseveral kilobases of flanking DNA (both at the 5′ and the 3′ ends) (see,for example, Thomas, K. R. and Capecchi, M. R. (1987) Cell 51: 503 for adescription of homologous recombination vectors). The vector isintroduced into a microorganism (e.g. by electroporation) and cells inwhich the introduced MP gene has homologously recombined with theendogenous MP gene are selected using methods known in the art.

In another embodiment, it is possible to produce recombinantmicroorganisms which contain selected systems which make possible aregulated expression of the introduced gene. The inclusion of an MP geneunder the control of the lac operon in a vector enables, for example, MPgene expression only in the presence of IPTG. These regulatory systemsare known in the art.

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, may be used for producing (i.e. expressing) an MPprotein. In addition, the invention provides methods for producing MPproteins by using the host cells of the invention. In one embodiment,the method comprises the cultivation of the host cell of the invention(into which a recombinant expression vector encoding an MP protein hasbeen introduced or in whose genome a gene encoding a wild-type ormodified MP protein has been introduced) in a suitable medium until theMP protein has been produced. In a further embodiment, the methodcomprises isolating the MP proteins from the medium or the host cell.

C. Uses and Methods of the Invention

The nucleic acid molecules, proteins, fusion proteins, primers, vectorsand host cells described herein may be used in one or more of thefollowing methods: identification of C. glutamicum and relatedorganisms, mapping of genomes of organisms related to C. glutamicum,identification and localization of C. glutamicum sequences of interest,evolutionary studies, determination of MP protein regions required forfunction, modulation of the activity of an MP protein; modulation of theactivity of an MP pathway; and modulation of the cellular production ofa compound of interest, such as a fine chemical. The MP nucleic acidmolecules of the invention have a multiplicity of uses. First, they maybe used for identifying an organism as Corynebacterium glutamicum orclose relatives thereof. They may also be used for identifying C.glutamicum or a relative thereof in a mixed population ofmicroorganisms. The invention provides the nucleic acid sequences of anumber of C. glutamicum genes. Probing the extracted genomic DNA of aculture of a uniform or mixed population of microorganisms understringent conditions with a probe which comprises a region of a C.glutamicum gene which is unique in this organism makes it possible todetermine whether said organism is present. Although Corynebacteriumglutamicum itself is nonpathogenic, it is related to pathogenic speciessuch as Corynebacterium diphtheriae. The detection of such an organismis of substantial clinical importance.

The nucleic acid and protein molecules of the invention may serve asmarkers for specific regions of the genome. This is useful not only formapping the genome but also for functional studies of C. glutamicumproteins. The genomic region to which a particular C. glutamicumDNA-binding protein binds may be identified, for example, by cleavingthe C. glutamicum genome and incubating the fragments with theDNA-binding protein. Those fragments which bind the protein mayadditionally be probed with the nucleic acid molecules of the invention,preferably by using readily detectable labels; binding of such a nucleicacid molecule to the genomic fragment makes it possible to locate thefragment on the map of the C. glutamicum genome, and carrying out thisprocess several times using different enzymes facilitates rapiddetermination of the nucleic acid sequence to which the protein binds.Moreover, the nucleic acid molecules of the invention may besufficiently homologous to the sequences of related species for thesenucleic acid molecules to serve as markers for constructing a genomicmap in related bacteria such as Brevibacterium lactofermentum.

The MP nucleic acid molecules of the invention are likewise suitable forevolutionary studies and protein structure studies. Many prokaryotic andeukaryotic cells utilize the metabolic processes in which the moleculesof the invention are involved; by comparing the sequences of the nucleicacid molecules of the invention with those sequences which encodesimilar enzymes from other organisms, it is possible to determine thedegree of evolutionary relationship of said organisms. Accordingly, sucha comparison makes it possible to determine which sequence regions areconserved and which are not, and this may be helpful in determiningthose regions of the protein which are essential for enzyme function.This type of determination is valuable for protein engineering studiesand may give an indication as to which protein can tolerate mutagenesiswithout losing its function.

Manipulation of the MP nucleic acid molecules of the invention may causethe production of MP proteins with functional differences to wild-typeMP proteins. These proteins can be improved with respect to theirefficiency or activity, can be present in the cell in larger amountsthan normal or can be weakened with respect to their efficiency oractivity.

These changes in activity may be such that the yield, production and/orefficiency of production of one or more fine chemicals from C.glutamicum are improved. By optimizing the activity of an MP proteinwhich activates transcription or translation of a gene encoding aprotein of the biosynthesis of a fine chemical of interest or byinfluencing or deleting the activity of an MP protein which repressestranscription or translation of such a gene, it is possible to increasethe activity or activity rate of this biosynthetic pathway, owing to thepresence of increased amounts of, for example, a limiting enzyme.Correspondingly, it is possible, by modifying the activity of an MPprotein such that it constitutively inactivates posttranslationally aprotein which is involved in the degradation pathway of a fine chemicalof interest or by modifying the activity of an MP protein such that itconstitutively represses transcription or translation of such a gene, toincrease the yield and/or production rate of said fine chemical from thecell, owing to the reduced degradation of the compound.

Modulating the activity of one or more MP proteins makes it possible toindirectly stimulate the production or to improve the production rate ofone or more fine chemicals from the cell, owing to the linkage ofvarious metabolic pathways. It is possible, for example, by increasingthe yield, production and/or efficiency of production by activating theexpression of one or more enzymes in lysine biosynthesis, to increasesimultaneously the expression of other compounds such as other aminoacids which the cell usually needs in larger quantities when largerquantities of lysine are required. It is also possible to modify themetabolic regulation in the entire cell such that the cell under theenvironmental conditions of a fermentation culture (in which the supplyof nutrients and oxygen may be poor and possibly toxic waste productsmay be present in large quantities in the environment) may have improvedgrowth or replication. Thus it is possible, for example, to improve thegrowth and propagation of the cells in culture, even if the growthconditions are suboptimal, by mutagenizing an MP protein whichsuppresses the synthesis of molecules required for cell membraneproduction in reaction to high levels of waste products in theextracellular medium (in order to block cell growth and cell division insuboptimal growth conditions) such that said protein is no longercapable of repressing said synthesis. Such increased growth or suchincreased viability should likewise increase the yields and/orproduction rate of a fine chemical of interest from a fermentativeculture, owing to the relatively large number of cells producing thiscompound in the culture.

The abovementioned strategies for the mutagenesis of MP proteins, whichought to increase the yields of a fine chemical of interest in C.glutamicum, are not intended to be limiting; variations of thesestrategies are quite obvious to the skilled worker. These strategies andthe mechanisms disclosed herein make it possible to use the nucleic acidand protein molecules of the invention in order to generate C.glutamicum or related bacterial strains expressing mutated MP nucleicacid and protein molecules so as to improve the yield, production and/orefficiency of production of a compound of interest. The compound ofinterest may be a natural C. glutamicum product which comprises the endproducts of the biosynthetic pathways and intermediates of naturallyoccurring metabolic pathways and also molecules which do not naturallyoccur in the C. glutamicum metabolism but are produced by a C.glutamicum strain of the invention.

The following examples, which are not to be understood as beinglimiting, further illustrate the present invention. The contents of allreferences, patent applications, patents and published patentapplications cited in this patent application are hereby incorporated byway of reference.

EXAMPLES Example 1 Preparation of Total Genomic DNA from Corynebacteriumglutamicum ATCC13032

A Corynebacterium glutamicum (ATCC 13032) culture was cultivated withvigorous shaking in BHI medium (Difco) at 30° C. overnight. The cellswere harvested by centrifugation, the supernatant was discarded and thecells were resuspended in 5 ml of buffer I (5% of the original culturevolume—all volumes stated have been calculated for a culture volume of100 ml). Composition of buffer I: 140.34 g/l sucrose, 2.46 g/lMgSO₄.7H₂O, 10 ml/l KH₂PO₄ solution (100 g/l, adjusted to pH 6.7 withKOH), 50 ml/l M12 concentrate (10 g/l (NH₄)₂SO₄, 1 g/l NaCl, 2 g/lMgSO₄.7H₂O, 0.2 g/l CaCl₂, 0.5 g/l yeast extract (Difco), 10 ml/l traceelement mixture (200 mg/l FeSO₄.H₂O, 10 mg/l ZnSO₄.7H₂O, 3 mg/lMnCl₂.4H₂O, 30 mg/l H₃BO₃, 20 mg/l CoCl₂.6H₂O, 1 mg/l NiCl₂.6H₂O, 3 mg/lNa₂MoO₄.2H₂O), 500 mg/l complexing agent (EDTA or citric acid), 100 ml/lvitamin mixture (0.2 ml/l biotin, 0.2 mg/l folic acid, 20 mg/lp-aminobenzoic acid, 20 mg/l riboflavin, 40 mg/l Ca pantothenate, 140mg/l nicotinic acid, 40 mg/l pyridoxal hydrochloride, 200 mg/lmyoinositol). Lysozyme was added to the suspension at a finalconcentration of 2.5 mg/ml. After incubation at 37° C. for approx. 4 h,the cell wall was degraded and the protoplasts obtained were harvestedby centrifugation. The pellet was washed once with 5 ml of buffer I andonce with 5 ml of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8). Thepellet was resuspended in 4 ml of TE buffer, and 0.5 ml of SDS solution(10%) and 0.5 ml of NaCl solution (5 M) were added. After addition ofproteinase K at a final concentration of 200 μg/ml, the suspension wasincubated at 37° C. for approx. 18 h. The DNA was purified viaextraction with phenol, phenol/chloroform/isoamyl alcohol andchloroform/isoamyl alcohol by means of standard methods. The DNA wasthen precipitated by addition of 1/50 volume of 3 M sodium acetate and 2volumes of ethanol, subsequent incubation at −20° C. for 30 min andcentrifugation at 12 000 rpm in a high-speed centrifuge using an SS34rotor (Sorvall) for 30 min. The DNA was dissolved in 1 ml of TE buffercontaining 20 μg/ml RNase A and dialyzed against 1000 ml of TE buffer at4° C. for at least 3 h. The buffer was exchanged 3 times during thisperiod. 0.4 ml of 2 M LiCl and 0.8 ml of ethanol were added to 0.4 mlaliquots of the dialyzed DNA solution. After incubation at −20° C. for30 min, the DNA was collected by centrifugation (13 000 rpm, BiofugeFresco, Heraeus, Hanau, Germany). The DNA pellet was dissolved in TEbuffer. It was possible to use DNA prepared by this method for allpurposes, including Southern blotting and constructing genomiclibraries.

Example 2 Construction of Genomic Corynebacterium glutamicum (ATCC13032)Banks in Escherichia coli

Starting from DNA prepared as described in example 1, cosmid and plasmidbanks were prepared according to known and well-established methods(see, for example, Sambrook, J. et al. (1989) “Molecular Cloning: ALaboratory Manual”. Cold Spring Harbor Laboratory Press or Ausubel, F.M. et al. (1994) “Current Protocols in Molecular Biology”, John Wiley &Sons).

It was possible to use any plasmid or cosmid. Particular preference wasgiven to using the plasmids pBR322 (Sutcliffe, J. G. (1979) Proc. Natl.Acad. Sci. USA, 75: 3737-3741); pACYC177 (Change & Cohen (1978) J.Bacteriol. 134: 1141-1156); pBS series plasmids (pBSSK+, pBSSK− andothers; Stratagene, La Jolla, USA) or cosmids such as SuperCos1(Stratagene, La Jolla, USA) or Lorist6 (Gibson, T. J. Rosenthal, A., andWaterson, R. H. (1987) Gene 53: 283-286).

Example 3 DNA Sequencing and Functional Computer Analysis

Genomic banks as described in example 2 were used for DNA sequencingaccording to standard methods, in particular the chain terminationmethod using ABI377 sequencers (see, for example, Fleischman, R. D. etal. (1995) “Whole-genome Random Sequencing and Assembly of HaemophilusInfluenzae Rd.”, Science 269; 496-512). Sequencing primers having thefollowing nucleotide sequences were used: 5′-GGAAACAGTATGACCATG-3′ (SEQID NO:143) or 5′-GTAAAACGACGGCCAGT-3′ (SEQ ID NO:144).

Example 4 In Vivo Mutagenesis

In vivo mutagenesis of Corynebacterium glutamicum may be carried out bypassing a plasmid (or other vector) DNA through E. coli or othermicroorganisms (e.g. Bacillus spp. or yeasts such as Saccharomycescerevisiae) which cannot maintain the integrity of their geneticinformation. Common mutator strains contain mutations in the genes forthe DNA repair system (e.g., mutHLS, mutD, mutT, etc., for comparisonsee Rupp, W. D. (1996) DNA repair mechanisms in Escherichia coli andSalmonella, pp. 2277-2294, ASM: Washington). These strains are known tothe skilled worker. The use of these strains is illustrated, forexample, in Greener, A. and Callahan, M. (1994) Strategies 7; 32-34.

Example 5 DNA Transfer Between Escherichia coli and Corynebacteriumglutamicum

A plurality of Corynebacterium and Brevibacterium species containendogenous plasmids (such as, for example, pHM1519 or pBL1) whichreplicate autonomously (for a review see, for example, Martin, J. F. etal. (1987) Biotechnology 5: 137-146). Shuttle vectors for Escherichiacoli and Corynebacterium glutamicum can be constructed readily by meansof standard vectors for E. coli (Sambrook, J. et al., (1989), “MolecularCloning: A Laboratory Manual”, Cold Spring Harbor Laboratory Press orAusubel, F. M. et al. (1994) “Current Protocols in Molecular Biology”,John Wiley & Sons), to which an origin of replication for and a suitablemarker from Corynebacterium glutamicum are added. Such origins ofreplication are preferably taken from endogenous plasmids which havebeen isolated from Corynebacterium and Brevibacterium species.Particularly useful transformation markers for these species are genesfor kanamycin resistance (such as those derived from the Tn5 or theTn903 transposon) or for chloramphenicol resistance (Winnacker, E. L.(1987) “From Genes to Clones—Introduction to Gene Technology”, VCH,Weinheim). There are numerous examples in the literature for preparing alarge multiplicity of shuttle vectors which are replicated in E. coliand C. glutamicum and which can be used for various purposes, includingthe overexpression of genes (see, for example, Yoshihama, M. et al.(1985) J. Bacteriol. 162: 591-597, Martin, J. F. et al., (1987)Biotechnology, 5: 137-146 and Eikmanns, B. J. et al. (1992) Gene 102:93-98).

Standard methods make it possible to clone a gene of interest into oneof the above-described shuttle vectors and to introduce such hybridvectors into Corynebacterium glutamicum strains. C. glutamicum can betransformed via protoplast transformation (Kastsumata, R. et al., (1984)J. Bacteriol. 159, 306-311), electroporation (Liebl, E. et al., (1989)FEMS Microbiol. Letters, 53: 399-303) and, in cases in which specificvectors are used, also via conjugation (as described, for example, inSchäfer, A., et al. (1990) J. Bacteriol. 172: 1663-1666). Likewise, itis possible to transfer the shuttle vectors for C. glutamicum to E. coliby preparing plasmid DNA from C. glutamicum (by means of standardmethods known in the art) and transforming it into E. coli. Thistransformation step can be carried out using standard methods butadvantageously an Mcr-deficient E. coli strain such as NM522 (Gough &Murray (1983) J. Mol. Biol. 166: 1-19) is used.

Example 6 Determination of the Expression of the Mutated Protein

The observations of the activity of a mutated protein in a transformedhost cell are based on the fact that the mutated protein is expressed ina similar manner and in similar quantity to the wild-type protein. Asuitable method for determining the amount of transcription of themutated gene (an indication of the amount of mRNA available fortranslation of the gene product) is to carry out a Northern blot (see,for example, Ausubel et al., (1988) Current Protocols in MolecularBiology, Wiley: New York), with a primer which is designed such that itbinds to the gene of interest being provided with a detectable (usuallyradioactive or chemiluminescent) label such that—when the total RNA of aculture of the organism is extracted, fractionated on a gel, transferredto a stable matrix and incubated with this probe—binding and bindingquantity of the probe indicate the presence and also the amount of mRNAfor said gene. This information is an indicator of the extent to whichthe mutated gene has been transcribed. Total cell RNA can be isolatedfrom Corynebacterium glutamicum by various methods known in the art, asdescribed in Bormann, E. R. et al., (1992) Mol. Microbiol. 6: 317-326.

The presence or the relative amount of protein translated from said mRNAcan be determined by using standard techniques such as Western blot(see, for example, Ausubel et al. (1988) “Current Protocols in MolecularBiology”, Wiley, New York). In this method, total cell proteins areextracted, separated by gel electrophoresis, transferred to a matrixsuch as nitrocellulose and incubated with a probe, for example anantibody, which binds specifically to the protein of interest. Thisprobe is usually provided with a chemiluminescent or calorimetric labelwhich can be readily detected. The presence and the observed amount oflabel indicate the presence and the amount of the desired mutant proteinin the cell.

Example 7 Growth of Genetically Modified Corynebacteriumglutamicum—Media and Cultivation Conditions

Genetically modified corynebacteria are cultivated in synthetic ornatural growth media. A number of different growth media forcorynebacteria are known and readily available (Lieb et al. (1989) Appl.Microbiol. Biotechnol. 32: 205-210; von der Osten et al. (1998)Biotechnology Letters 11: 11-16; Patent DE 4 120 867; Liebl (1992) “TheGenus Corynebacterium”, in: The Procaryotes, Vol. II, Balows, A., etal., editors Springer-Verlag). These media are composed of one or morecarbon sources, nitrogen sources, inorganic salts, vitamins and traceelements. Preferred carbon sources are sugars such as mono-, di- orpolysaccharides. Examples of very good carbon sources are glucose,fructose, mannose, galactose, ribose, sorbose, ribulose, lactose,maltose, sucrose, raffinose, starch and cellulose. Sugars may also beadded to the media via complex compounds such as molasses or otherbyproducts from sugar refining. It may also be advantageous to addmixtures of various carbon sources. Other possible carbon sources arealcohols and organic acids, such as methanol, ethanol, acetic acid orlactic acid. Nitrogen sources are usually organic or inorganic nitrogencompounds or materials containing these compounds. Examples of nitrogensources include ammonia gas and ammonium salts such as NH₄Cl or(NH₄)₂SO₄, NH₄OH, nitrates, urea, amino acids and complex nitrogensources such as corn steep liquor, soya meal, soya protein, yeastextracts, meat extracts and others.

Inorganic salt compounds which may be present in the media include thechloride, phosphorus or sulfate salts of calcium, magnesium, sodium,cobalt, molybdenum, potassium, manganese, zinc, copper and iron.Chelating agents may be added to the medium in order to keep the metalions in solution. Particularly suitable chelating agents includedihydroxyphenols such as catechol or protocatechuate and organic acidssuch as citric acid. The media usually also contain other growth factorssuch as vitamins or growth promoters, examples of which include biotin,riboflavin, thiamin, folic acid, nicotinic acid, pantothenate andpyridoxine. Growth factors and salts are frequently derived from complexmedia components such as yeast extract, molasses, corn steep liquor andthe like. The exact composition of the media heavily depends on theparticular experiment and is decided upon individually for each case.Information on the optimization of media can be found in the textbook“Applied Microbiol. Physiology, A Practical Approach” (editors P. M.Rhodes, P. F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3).Growth media can also be obtained from commercial suppliers, for exampleStandard 1 (Merck) or BHI (brain heart infusion, DIFCO) and the like.

All media components are sterilized, either by heat (20 min at 1.5 barand 121° C.) or by sterile filtration. The components may be sterilizedeither together or, if required, separately. All media components may bepresent at the start of the cultivation or added continuously orbatchwise, as desired.

The cultivation conditions are defined separately for each experiment.The temperature should be between 15° C. and 45° C. and may be keptconstant or may be altered during the experiment. The pH of the mediumshould be in the range from 5 to 8.5, preferably around 7.0, and may bemaintained by adding buffers to the media. An example of a buffer forthis purpose is a potassium phosphate buffer. Synthetic buffers such asMOPS, HEPES, ACES, etc. may be used alternatively or simultaneously.Addition of NaOH or NH₄OH can also keep the pH constant duringcultivation. If complex media components such as yeast extract are used,the demand for additional buffers decreases, since many complexcompounds have a high buffer capacity. In the case of using a fermenterfor cultivating microorganisms, the pH may also be regulated usinggaseous ammonia.

The incubation period is usually in a range from several hours toseveral days. This time is selected such that the maximum amount ofproduct accumulates in the broth. The growth experiments disclosed maybe carried out in a multiplicity of containers such as microtiterplates, glass tubes, glass flasks or glass or metal fermenters ofdifferent sizes. For the screening of a large number of clones, themicroorganisms should be grown in microtiter plates, glass tubes orshaker flasks either with or without baffles. Preference is given tousing 100 ml shaker flasks which are filled with 10% (based on volume)of the required growth medium. The flasks should be shaken on an orbitalshaker (amplitude 25 mm) at a speed in the range of 100-300 rpm. Lossesdue to evaporation can be reduced by maintaining a humid atmosphere;alternatively, the losses due to evaporation should be correctedmathematically.

If genetically modified clones are investigated, an unmodified controlclone or a control clone containing the basic plasmid without insertshould also be assayed. The medium is inoculated to an OD₆₀₀ of 0.5-1.5,with cells being used which have been grown on agar plates such as CMplates (10 g/l glucose, 2.5 g/l NaCl, 2 μl urea, 10 g/l polypeptone, 5g/l yeast extract, 5 g/l meat extract, 22 μl agar pH 6.8 with 2 M NaOH)which have been incubated at 30° C. The media are inoculated either byintroducing a saline solution of C. glutamicum cells from CM plates orby adding a liquid preculture of said bacterium.

Example 8 In vitro Analysis of the Function of Mutated Proteins

The determination of the activities and kinetics of enzymes is wellknown in the art. Experiments for determining the activity of aparticular modified enzyme must be adapted to the specific activity ofthe wild-type enzyme, and this is within the capabilities of the skilledworker. Overviews regarding enzymes in general and also specific detailsconcerning the structure, kinetics, principles, methods, applicationsand examples of the determination of many enzyme activities can befound, for example, in the following references: Dixon, M., and Webb, E.C: (1979) Enzymes, Longmans, London; Fersht (1985) Enzyme Structure andMechanism, Freeman, New York; Walsh (1979) Enzymatic ReactionMechanisms. Freeman, San Francisco; Price, N.C., Stevens, L. (1982)Fundamentals of Enzymology. Oxford Univ. Press: Oxford; Boyer, P. D:editors (1983) The Enzymes, 3rd edition, Academic Press, New York;Bisswanger, H. (1994) Enzymkinetik, 2nd edition VCH, Weinheim (ISBN3527300325); Bergmeyer, H. U., Bergmeyer, J., Graβl, M. editors(1983-1986) Methods of Enzymatic Analysis, 3rd edition, Vol. I-XII,Verlag Chemie: Weinheim; and Ullmann's Encyclopedia of IndustrialChemistry (1987) Vol. A9, “Enzymes”, VCH, Weinheim, pp. 352-363.

The activities of proteins binding to DNA can be measured by manywell-established methods such as DNA bandshift assays (which are alsoreferred to as gel retardation assays). The action of these proteins onthe expression of other molecules can be measured using reporter geneassays (as described in Kolmar, H. et al., (1995) EMBO J. 14: 3895-3904and in references therein). Reporter gene assay systems are well knownand established for applications in prokaryotic and eukaryotic cells,with enzymes such as beta-galactosidase, green fluorescent protein andseveral other enzymes being used.

The activity of membrane transport proteins can be determined accordingto the techniques described in Gennis, R. B. (1989) “Pores, Channels andTransporters”, in Biomembranes, Molecular Structure and Function,Springer: Heidelberg, pp. 85-137; 199-234; and 270-322.

Example 9 Analysis of the Influence of Mutated Protein on the Productionof the Product of Interest

The effect of the genetic modification in C. glutamicum on theproduction of a compound of interest (such as an amino acid) can bedetermined by growing the modified microorganisms under suitableconditions (such as those described above) and testing the medium and/orthe cellular components for increased production of the product ofinterest (i.e. an amino acid). Such analytical techniques are well knownto the skilled worker and include spectroscopy, thin-layerchromatography, various types of staining methods, enzymic andmicrobiological methods and analytical chromatography such as highperformance liquid chromatography (see, for example, Ullmann,Encyclopedia of Industrial Chemistry, Vol. A2, pp. 89-90 and pp.443-613, VCH: Weinheim (1985); Fallon, A., et al., (1987) “Applicationsof HPLC in Biochemistry” in: Laboratory Techniques in Biochemistry andMolecular Biology, Vol. 17; Rehm et al. (1993) Biotechnology, Vol. 3,Chapter III: “Product recovery and purification”, pp. 469-714, VCH:Weinheim; Belter, P. A. et al. (1988) Bioseparations: downstreamprocessing for Biotechnology, John Wiley and Sons; Kennedy, J. F. andCabral, J. M. S. (1992) Recovery processes for biological Materials,John Wiley and Sons; Shaeiwitz, J. A. and Henry, J. D. (1988)Biochemical Separations, in Ullmann's Encyclopedia of IndustrialChemistry, Vol. B3; Chapter 11, pp. 1-27, VCH: Weinheim; and Dechow, F.J. (1989) Separation and purification techniques in biotechnology, NoyesPublications).

In addition to measuring the end product of the fermentation, it islikewise possible to analyze other components of the metabolic pathways,which are used for producing the compound of interest, such asintermediates and byproducts, in order to determine the overallproductivity of the organism, the yield and/or the efficiency ofproduction of the compound. The analytical methods include measuring theamounts of nutrients in the medium (for example sugars, hydrocarbons,nitrogen sources, phosphate and other ions), measuring biomasscomposition and growth, analyzing the production of common metabolitesfrom biosynthetic pathways and measuring gases generated duringfermentation. Standard methods for these measurements are described inApplied Microbial Physiology; A Practical Approach, P. M. Rhodes and P.F. Stanbury, editors IRL Press, pp. 103-129; 131-163 and 165-192 (ISBN:0199635773) and the references therein.

Example 10 Purification of the Product of Interest from a C. glutamicumCulture

The product of interest may be obtained from C. glutamicum cells or fromthe supernatant of the above-described culture by various methods knownin the art. If the product of interest is not secreted by the cells, thecells may be harvested from the culture by slow centrifugation, and thecells may be lyzed by standard techniques such as mechanical force orultrasonication. The cell debris is removed by centrifugation and thesupernatant fraction which contains the soluble proteins is obtained forfurther purification of the compound of interest. If the product issecreted by the C. glutamicum cells, the cells are removed from theculture by slow centrifugation and the supernatant fraction is retainedfor further purification.

The supernatant fraction from both purification methods is subjected tochromatography using a suitable resin, and either the molecule ofinterest is retained on the chromatography resin while many contaminantsin the sample are not or the contaminants remain on the resin while thesample does not. If necessary, these chromatography steps can berepeated using the same or different chromatography resins. The skilledworker is familiar with the selection of suitable chromatography resinsand the most effective application for a particular molecule to bepurified. The purified product may be concentrated by filtration orultrafiltration and stored at a temperature at which product stabilityis highest.

In the art, many purification methods are known which are not limited tothe above purification method and which are described, for example, inBailey, J. E. & Ollis, D. F. Biochemical Engineering Fundamentals,McGraw-Hill: New York (1986).

The identity and purity of the isolated compounds can be determined bystandard techniques of the art. These techniques comprise highperformance liquid chromatography (HPLC), spectroscopic methods,coloring methods, thin-layer chromatography, NIRS, enzyme assays ormicrobiological assays. These analytical methods are compiled in: Pateket al. (1994) Appl. Environ. Microbiol. 60: 133-140; Malakhova et al.(1996) Biotekhnologiya 11: 27-32; and Schmidt et al. (1998) BioprocessEngineer. 19: 67-70. Ulmann's Encyclopedia of Industrial Chemistry(1996) Vol. A27, VCH: Weinheim, pp. 89-90, pp. 521-540, pp. 540-547, pp.559-566, pp. 575-581 and pp. 581-587; Michal, G. (1999) BiochemicalPathways: An Atlas of Biochemistry and Molecular Biology, John Wiley andSons; Fallon, A. et al. (1987) Applications of HPLC in Biochemistry in:Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 17.

Equivalents

The skilled worker knows, or can identify by using simply routinemethods, a large number of equivalents of the specific embodiments ofthe invention. These equivalents are intended to be included in thepatent claims below.

The information in table 1 and table 2 is to be understood as follows:

In column 1, “DNA ID”, the relevant number refers in each case to theSEQ ID NO of the enclosed sequence listing. Consequently, “5” in column“DNA ID” is a reference to SEQ ID NO:5.

In column 2, “AA ID”, the relevant number refers in each case to the SEQID NO of the enclosed sequence listing. Consequently, “6” in column “AAID” is a reference to SEQ ID NO:6.

In column 3, “Identification”, an unambiguous internal name for eachsequence is listed.

In column 4, “AA position”, the relevant number refers in each case tothe amino acid position of the polypeptide sequence “AA ID” in the samerow. Consequently, “26” in column “M position” is amino acid position 26of the polypeptide sequence indicated accordingly. Position countingstarts at the N terminus with +1.

In column 5, “AA wild type”, the relevant letter refers in each case tothe amino acid, displayed in the one-letter code, at the position in thecorresponding wild-type strain, which is indicated in column 4.

In column 6, “AA mutant”, the relevant letter refers in each case to theamino acid, displayed in the one-letter code, at the position in thecorresponding mutant strain, which is indicated in column 4.

In column 7, “Function”, the physiological function of the correspondingpolypeptide sequence is listed.

Columns 4, 5 and 6 describe at least one mutation, in the case of somesequences also several mutations, for an MP protein with a particularfunction (column 7) and a particular starting amino acid sequence(column 2). Said several mutations always refer to the nearest startingamino acid sequence, in each case listed at the top (column 2). The term“at least one of the amino acid positions” of a particular amino acidsequence preferably means at least one of the mutations described forthis amino acid sequence in column 4, 5 and 6.

One-Letter Code of the Proteinogenic Amino Acids:

-   A Alanine-   C Cysteine-   D Aspartic acid-   E Glutamic acid-   F Phenylalanine-   G Glycine-   H Histidine-   I Isoleucine-   K Lysine-   L Leucine-   M Methionine-   N Asparagine-   P Proline-   Q Glutamine-   R Arginine-   S Serine-   T Threonine-   V Valine-   W Tryptophan-   Y Tyrosine

TABLE 1 MP proteins of the invention Column 1 Column 2 Column 4 Column 5Column 6 DNA AA Column 3 AA AA AA Column 7 ID ID Identification positionwild type mutant Function 1 2 RXA00149K 85 T S Methylmalonyl-CoA mutase166 T A Methylmalonyl-CoA mutase 174 F P Methylmalonyl-CoA mutase 350 DG Methylmalonyl-CoA mutase 436 A T Methylmalonyl-CoA mutase 538 A SMethylmalonyl-CoA mutase 3 4 RXA00157E 26 T A Invasin 1 247 S T Invasin1 255 G E Invasin 1 336 T A Invasin 1 342 A T Invasin 1 395 A V Invasin1 448 T A Invasin 1 456 S N Invasin 1 457 N D Invasin 1 5 6 RXA00157K217 S T Invasin 1 225 G E Invasin 1 306 T A Invasin 1 312 A T Invasin 1365 A V Invasin 1 418 T A Invasin 1 5 6 RXA00157K 426 S N Invasin 1 427N D Invasin 1 60 V I Invasin 1 129 G R Invasin 1 419 G D Invasin 1 424 SF Invasin 1 560 S F Invasin 1 7 8 RXA00330W 69 G R Threonine synthase 910 RXA00683W 95 D E PEP synthase 119 V I PEP synthase 348 I T PEPsynthase 11 12 RXA00783E 2 K E Succinyl-CoA synthetase 13 D GSuccinyl-CoA synthetase 66 G E Succinyl-CoA synthetase 112 N DSuccinyl-CoA synthetase 171 T I Succinyl-CoA synthetase 189 T ASuccinyl-CoA synthetase 224 D G Succinyl-CoA synthetase 334 Q HSuccinyl-CoA synthetase 364 G D Succinyl-CoA synthetase 13 14 RXA00783K40 G E Succinyl-CoA synthetase 86 N D Succinyl-CoA synthetase 145 T ISuccinyl-CoA synthetase 163 T A Succinyl-CoA synthetase 198 D GSuccinyl-CoA synthetase 13 14 RXA00783K 308 Q H Succinyl-CoA synthetase338 G D Succinyl-CoA synthetase 18 P S Succinyl-CoA synthetase 166 A TSuccinyl-CoA synthetase 15 16 RXA00969K 104 V I Homoserine dehydrogenase116 T I Homoserine dehydrogenase 17 18 RCGL01443K 11 S A Transcriptionalregulator 140 N K Transcriptional regulator 228 F M Transcriptionalregulator 19 20 RXA00999K 329 V M 6-Phosphogluconate dehydrogenase 21 22RXA01093W 329 S A Pyruvate kinase 436 A T Pyruvate kinase 23 24RXA01244W 178 D E Phosphoenolpyruvate protein phosphotransferase 384 E KPhosphoenolpyruvate protein phosphotransferase 25 26 RXA01434E 61 P LVirulence Factor MVIN 515 N K Virulence Factor MVIN 580 F I VirulenceFactor MVIN 581 V I Virulence Factor MVIN 607 L M Virulence Factor MVIN626 E Q Virulence Factor MVIN 640 A S Virulence Factor MVIN 743 H YVirulence Factor MVIN 1080 V M Virulence Factor MVIN 1088 R H VirulenceFactor MVIN 27 28 RXA01434K 35 P L Virulence Factor MVIN 489 N KVirulence Factor MVIN 554 F I Virulence Factor MVIN 555 V I VirulenceFactor MVIN 581 L M Virulence Factor MVIN 600 E Q Virulence Factor MVIN614 A S Virulence Factor MVIN 717 H Y Virulence Factor MVIN 1054 V MVirulence Factor MVIN 1062 R H Virulence Factor MVIN 931 S F VirulenceFactor MVIN 986 P S Virulence Factor MVIN 29 30 RXA02056E 4 T I2-Oxoglutarate dehydrogenase 64 T A 2-Oxoglutarate dehydrogenase 102 D A2-Oxoglutarate dehydrogenase 258 A T 2-Oxoglutarate dehydrogenase 398 AT 2-Oxoglutarate dehydrogenase 987 I V 2-Oxoglutarate dehydrogenase 1113N D 2-Oxoglutarate dehydrogenase 31 32 RXA02056K 20 T I 2-Oxoglutaratedehydrogenase 80 T A 2-Oxoglutarate dehydrogenase 118 D A 2-Oxoglutaratedehydrogenase 274 A T 2-Oxoglutarate dehydrogenase 414 A T2-Oxoglutarate dehydrogenase 1075 P L 2-Oxoglutarate dehydrogenase 31 32RXA02056K 1137 A V 2-Oxoglutarate dehydrogenase 33 34 RXA02063E 394 Q RGlucose 1-phosphate adenylyl tranferase 35 36 RXA02063W 386 Q R Glucose1-phosphate adenylyl tranferase 129 G D Glucose 1-phosphate adenylyltranferase 37 38 RXA02082K 156 E D SMC2 316 A T SMC2 349 T A SMC2 682 VA SMC2 832 Q H SMC2 39 40 RXA02016W 20 T S Nitrate reductase 49 A TNitrate reductase 58 N H Nitrate reductase 59 T A Nitrate reductase 131S I Nitrate reductase 166 N D Nitrate reductase 317 A S Nitratereductase 41 42 RXA02149W 63 G D Hexokinase 99 N D Hexokinase 43 44RXA02206E 114 A S Oxidoreductase 126 A V Oxidoreductase 45 46 RXA02206K74 A S Oxidoreductase 86 A V Oxidoreductase 33 G D Oxidoreductase 74 A TOxidoreductase 274 R C Oxidoreductase 47 48 RXA02299W 65 G R Aspartate1-decarboxylase 49 50 RXA02390W 85 I M Threonine efflux protein 161 F IThreonine efflux protein 51 52 RXA02399E 109 S F Isocitrate lyase 155 TS Isocitrate lyase 290 A T Isocitrate lyase 53 54 RXA02399W 93 S FIsocitrate lyase 139 T S Isocitrate lyase 274 A T Isocitrate lyase 332 AT Isocitrate lyase 55 56 RXA02404K 80 M T Malate synthase 96 E D Malatesynthase 221 I V Malate synthase 232 E D Malate synthase 246 A V Malatesynthase 300 D N Malate synthase 326 I T Malate synthase 371 G D Malatesynthase 57 58 RXA02591W 586 L F PEP carboxykinase 59 60 RXA02735E 73 GS 6-Phosphogluconolactonase 61 62 RXA02735W 33 G S6-Phosphogluconolactonase 63 64 RXA02736W 107 Y H OPCA 219 K N OPCA 233P S OPCA 261 Y H OPCA 65 66 RXA02737W 8 S T Glucose 6-phosphatedehydrogenase 321 G S Glucose 6-phosphate dehydrogenase 67 68 RXA02738W242 K M Transaldolase 69 70 RXA02739K 75 N D Transketolase 332 A TTransketolase 556 V I Transketolase 71 72 RXA02910K 108 L ITranscriptional regulator 152 E G Transcriptional regulator 183 D NTranscriptional regulator 73 74 RXA02910E 111 L I Transcriptionalregulator 155 E G Transcriptional regulator 75 76 RXA03083W 140 V IDihadrolipoamide dehydrogenase 77 78 RXA03802K 149 V A Transcriptionalregulator 157 N K Transcriptional regulator 158 C S Transcriptionalregulator 190 S G Transcriptional regulator 79 80 RXA04031K 7 S LPyruvate carboxylase 639 S T Pyruvate carboxylase 1008 R H Pyruvatecarboxylase 1059 S P Pyruvate carboxylase 1120 D E Pyruvate carboxylase81 82 RXA04073K 39 E K unknown function 83 84 RXA04174K 75 D N Aconitase790 G S Aconitase 892 T I Aconitase 85 86 RXA02175W 8 A T Citratesynthase 283 S N Citrate synthase 291 E A Citrate synthase 420 N KCitrate synthase 87 88 RXA02175E 14 A T Citrate synthase 89 90 RXA04186K348 K E 5-Methyltetrahydrofolate homocysteine methyltransferase 474 E D5-Methyltetrahydrofolate homocysteine methyltransferase 731 E K5-Methyltetrahydrofolate homocysteine methyltransferase 747 E K5-Methyltetrahydrofolate homocysteine methyltransferase 91 92 RXA00098E54 V A Glucose 6-P isomerase 146 A P Glucose 6-P isomerase 93 94RXA00241K 5 S T Lysine-specific permease 501 F L Lysine specificpermease 95 96 RXA00517E 110 H Y Malate dehydrogenase 229 A S Malatedehydrogenase 236 V I Malate dehydrogenase 272 D E Malate dehydrogenase306 G T Malate dehydrogenase 307 E Q Malate dehydrogenase 326 S L Malatedehydrogenase 97 98 RXA00517W 95 H Y Malate dehydrogenase 97 98RXA00517W 214 A S Malate dehydrogenase 221 V I Malate dehydrogenase 257D E Malate dehydrogenase 291 G T Malate dehydrogenase 292 E Q Malatedehydrogenase 311 S L Malate dehydrogenase 99 100 RXA00519W 111 N TIsocitrate dehydrogenase 120 I T Isocitrate dehydrogenase 306 D NIsocitrate dehydrogenase 321 D E Isocitrate dehydrogenase 101 102RXA00533E 50 D G Aspartate semialdehyde dehydrogenase 218 R H Aspartatesemialdehyde dehydrogenase 256 D E Aspartate semialdehyde dehydrogenase269 K E Aspartate semialdehyde dehydrogenase 103 104 RXA00533K 234 R HAspartate semialdehyde dehydrogenase 105 106 RXA00782W 181 I V Succinatethiokinase 107 108 RXA00822K 6 E D Regulatory protein 109 110 RXA00975K355 G D Arginyl-tRNA synthetase 513 V A Arginyl-tRNA synthetase 540 H RArginyl-tRNA synthetase 111 112 RXA01311E 92 K N Succinate dehydrogenase113 114 RXA01311K 122 K N Succinate dehydrogenase 115 116 RXA01312E 328E V Succinate dehydrogenase 329 P K Succinate dehydrogenase 330 N GSuccinate dehydrogenase 115 116 RXA01312E 332 N D Succinatedehydrogenase 496 S A Succinate dehydrogenase 500 S A Succinatedehydrogenase 507 Q V Succinate dehydrogenase 508 A Q Succinatedehydrogenase 511 A E Succinate dehydrogenase 557 E K Succinatedehydrogenase 564 N D Succinate dehydrogenase 571 D E Succinatedehydrogenase 117 118 RXA01312K 280 E V Succinate dehydrogenase 281 P KSuccinate dehydrogenase 282 N G Succinate dehydrogenase 284 N DSuccinate dehydrogenase 448 S A Succinate dehydrogenase 452 S ASuccinate dehydrogenase 459 Q V Succinate dehydrogenase 460 A QSuccinate dehydrogenase 463 A E Succinate dehydrogenase 509 E KSuccinate dehydrogenase 516 N D Succinate dehydrogenase 523 D ESuccinate dehydrogenase 119 120 RXA01350E 109 P S Malate dehydrogenase196 S F Malate dehydrogenase 315 A G Malate dehydrogenase 121 122RXA01350W 68 P S Malate dehydrogenase 121 122 RXA01350W 155 S F Malatedehydrogenase 274 A G Malate dehydrogenase 272 T I Malate dehydrogenase123 124 RXA01393W 5 Q H Regulator IysG 125 126 RXA02021W 45 E V2,3,4,5-Tetrahydropyridine 2-carboxylate N- succinyltransferase 68 Q H2,3,4,5-Tetrahydropyridine 2-carboxylate N- succinyltransferase 122 D N2,3,4,5-Tetrahydropyridine 2-carboxylate N- succinyltransferase 275 G E2,3,4,5-Tetrahydropyridine 2-carboxylate N- succinyltransferase 283 A D2,3,4,5-Tetrahydropyridine 2-carboxylate N- succinyltransferase 127 128RXA02022E 141 T I 2,3,4,5-Tetrahydropyridine 2-carboxylate N-succinyltransferase 129 130 RXA02022K 114 T I 2,3,4,5-Tetrahydropyridine2-carboxylate N- succinyltransferase 131 132 RXA02139K 319 A T Asparaginsynthetase 576 T A Asparagin synthetase 133 134 RXA02157W 36 N KAcetylornithine aminotransferase/N-Succinyl-L,L- DAP aminotransferase309 A T Acetylornithine aminotransferase/N-Succinyl-L,L- DAPaminotransferase 135 136 RXA02229K 158 G S Diaminopimelate epimerase 137138 RXA02259E 184 H Y PEP carboxylase 263 G D PEP carboxylase 851 T RPEP carboxylase 139 140 RXA02259K 162 H Y PEP carboxylase 141 142RXA00970W 103 S A Homoserine kinase 190 T A Homoserine kinase

TABLE 2 Excepted MP proteins Column 1 Column 2 Column 4 Column 5 Column6 DNA AA Column 3 AA AA AA Column7 ID ID Identification position wildtype mutant Function 3 4 RXA00157E 90 V I Invasin 1 159 G R Invasin 1449 G D Invasin 1 454 S F Invasin 1 590 S F Invasin 1 11 12 RXA00783E 66G E Succinyl-CoA synthetase 44 P S Succinyl-CoA synthetase 192 A TSuccinyl-CoA synthetase 25 26 RXA01434E 957 S F Virulence Factor MVIN1012 P S Virulence Factor MVIN 29 30 RXA02056E 1059 P L 2-Oxoglutaratedehydrogenase 1121 A V 2-Oxoglutarate dehydrogenase 33 34 RXA02063E 137G D Glucose 1-phosphate adenylyl tranferase 43 44 RXA02206E 73 G DOxidoreductase 114 A T Oxidoreductase 314 R C Oxidoreductase 51 52RXA02399E 348 A T Isocitrate lyase 73 74 RXA02910E 186 D NTranscriptional regulator 119 120 RXA01350E 313 T I Malate dehydrogenase

1. An isolated nucleic acid molecule encoding a transketolase selectedfrom the group consisting of: a) an isolated nucleic acid moleculeencoding the amino acid sequence set forth in SEQ ID NO: 70, wherein (i)the amino acid residue at position 75 of SEQ ID NO: 70 is any amino acidexcept asparagine, (ii) the amino acid residue at position 332 of SEQ IDNO: 70 is any amino acid except alanine, or (iii) the amino acid residueat position 556 of SEQ ID NO: 70 is any amino acid except valine, or afull complement thereof; and b) an isolated nucleic acid moleculecomprising the nucleotide sequence set forth in SEQ ID NO: 69, whereinthe nucleic acid molecule comprises one or more nucleic acidmodifications at (i) nucleotide residues 223-225 of SEQ ID NO: 69 suchthat nucleotide residues 223-225 of SEQ ID NO: 69 encode any amino acidexcept asparagine, (ii) nucleotide residues 994-996 of SEQ ID NO: 69such that nucleotide residues 994-996 of SEQ ID NO: 69 encode any aminoacid except alanine, or (iii) nucleotide residues 1666-1668 of SEQ IDNO: 69 such that nucleotide residues 1666-1668 of SEQ ID NO: 69 encodeany amino acid except valine, or a full complement thereof.
 2. Anisolated nucleic acid construct, comprising at least one nucleic acidmolecule of claim
 1. 3. The nucleic acid construct of claim 2,comprising at least one promoter and a terminator.
 4. A method forpreparing an isolated transformed host cell comprising introducing intothe host cell at least one of a) the nucleic acid molecule of claim 1;or b) a promoter which is heterologous to an endogenous nucleic acidmolecule and which enables the expression of said endogenous nucleicacid molecule, wherein said nucleic acid molecule is selected from thegroup consisting of: i) an isolated nucleic acid molecule encoding theamino acid sequence set forth in SEQ ID NO: 70, wherein either (a) theamino acid residue at position 75 of SEQ ID NO: 70 is any amino acidexcept asparagine, (b) the amino acid residue at position 332 of SEQ IDNO: 70 is any amino acid except alanine, or (c) the amino acid residueat position 556 of SEQ ID NO: 70 is any amino acid except valine, or afull complement thereof; and ii) an isolated nucleic acid moleculecomprising the nucleotide sequence set forth in SEQ ID NO: 69, whereinthe nucleic acid molecule comprises one or more nucleic acidmodifications at either (a) nucleotide residues 223-225 of SEQ ID NO: 69such that nucleotide residues 223-225 of SEQ ID NO: 69 encode any aminoacid except asparagine, (b) nucleotide residues 994-996 of SEQ ID NO: 69such that nucleotide residues 994-996 of SEQ ID NO: 69 encode any aminoacid except alanine, or (c) nucleotide residues 1666-1668 of SEQ ID NO:69 such that nucleotide residues 1666-1668 of SEQ ID NO: 69 encode anyamino acid except valine, or a full complement thereof.
 5. The method ofclaim 4, wherein the nucleic acid molecule of a), or the promoter of b),is introduced into a replicating plasmid or is integrated chromosomally.6. The method of claim 4, wherein the promoter of b) is functionallylinked to the endogenous nucleic acid molecule.
 7. An isolatedtransformed host cell, transformed with at least one of a) the nucleicacid molecule of claim 1; or c) a promoter which is heterologous to anendogenous nucleic acid molecule and which enables the expression ofsaid endogenous nucleic acid molecule, wherein said nucleic acidmolecule is selected from the group consisting of: i) an isolatednucleic acid molecule encoding the amino acid sequence set forth in SEQID NO: 70, wherein either (a) the amino acid residue at position 75 ofSEQ ID NO: 70 is any amino acid except asparagine, (b) the amino acidresidue at position 332 of SEQ ID NO: 70 is any amino acid exceptalanine, or (c) the amino acid residue at position 556 of SEQ ID NO: 70is any amino acid except valine, or a full complement thereof; and ii)an isolated nucleic acid molecule comprising the nucleotide sequence setforth in SEQ ID NO: 69, wherein the nucleic acid molecule comprises oneor more nucleic acid modifications at either (a) nucleotide residues223-225 of SEQ ID NO: 69 such that nucleotide residues 223-225 of SEQ IDNO: 69 encode any amino acid except asparagine, (b) nucleotide residues994-996 of SEQ ID NO: 69 such that nucleotide residues 994-996 of SEQ IDNO: 69 encode any amino acid except alanine, or (c) nucleotide residues1666-1668 of SEQ ID NO: 69 such that nucleotide residues 1666-1668 ofSEQ ID NO: 69 encode any amino acid except valine, or a full complementthereof.
 8. The isolated transformed host cell of claim 7, wherein theorganism is capable of producing an amino acid.
 9. A method for thepreparation of an amino acid, comprising culturing the isolatedtransformed host cell of claim 7 in a culturing medium under conditionssuitable for the preparation of an amino acid.
 10. The method of claim9, wherein the amino acid is isolated from the isolated transformed hostcell and/or the culturing medium.
 11. The method of claim 9, wherein theisolated transformed host cell is a microorganism.
 12. The method ofclaim 11, wherein the microorganism belongs to the bacterial genusCorynebacterium or Brevibacterium.
 13. The method of claim 9, whereinthe amino acid is selected from the group consisting of L-lysine,L-threonine and L-methionine.
 14. The isolated nucleic acid molecule ofclaim 1(a), wherein the amino acid residue at position 75 of SEQ ID NO:70 is aspartic acid, the amino acid residue at position 332 of SEQ IDNO: 70 is threonine, or the amino acid residue at position 556 of SEQ IDNO: 70 is isoleucine.
 15. The isolated nucleic acid molecule of claim1(b) wherein nucleotide residues 223-225 of SEQ ID NO: 69 encodeaspartic acid, nucleotide residues 994-996 of SEQ ID NO: 69 encodethreonine, or nucleotide residues 1666-1668 of SEQ ID NO: 69 encodeisoleucine.